Method for the assembly of a polynucleic acid sequence

ABSTRACT

Provided herein are methods for the assembly of a polynucleic acid sequence that is at least partially carried out on a microfluidic device; methods for the preparation of a library of polynucleic acid sequences; microfluidic devices; methods for designing nucleic acid sequences; methods for planning the assembly of a polynucleic acid sequence from a plurality of nucleic acid sequences; systems comprising components for carrying out these methods; computer programs which, when run on a computer, implements these methods; and computer readable medium or carrier signals encoding such a computer program.

FIELD OF THE INVENTION

The present invention relates to a method for the assembly of apolynucleic acid sequence that is at least partially carried out on amicrofluidic device. The present invention also relates to a method forthe preparation of a library of polynucleic acid sequences, amicrofluidic device, a method for designing nucleic acid sequencessuitable for use in a method of the invention, a method for planning theassembly of a polynucleic acid sequence from a plurality of nucleic acidsequences to be carried out by a method of the invention, a systemcomprising means for carrying out either of said methods, a computerprogram which, when run on a computer, implements either of said methodsand a computer readable medium or carrier signal encoding such acomputer program.

BACKGROUND TO THE INVENTION

Synthetic biology brings together the disciplines of engineering,biology and bioinformatics. Its focus is to make the engineering ofbiology easier and more predictable. Synthetic biology could improveproduction methods in a wide variety of markets such as biofuels andenergy, environmentally friendly chemicals, drug development and newmaterial fabrication. It is estimated that the synthetic biologyresearch market could be worth over $1.5 billion by 2013.

Microengineering capabilities can help to address some of the mostsignificant challenges faced in the field. For example, the use ofintegrated microfluidic systems provides biologists with a powerfulplatform for advancing synthetic biology.

Microfluidics deals with the manipulation of minute amounts of fluid(usually micro or nanoliters) within microchannels. Biological targetscan be transported in these channels for diverse manipulation. Theadvantages of microfluidic systems over conventional systems includereagent consumption reduction, waste reduction, cost-effectiveness andportability. Additionally, microfluidic technologies have the potentialto achieve high-throughput, highly parallel biological operations. Manychallenges are encountered in the fabrication of highly integratedmicrofluidic systems, such as the assembly of different materials andthe integration of active components.

Kong et al, Nucleic Acids Res. 2007; 35(8): e61; Epub 2007 Apr. 2 andWO2007/137242 describes parallel gene synthesis in a microfluidicdevice. Kong et al report the fabrication of a multi-chambermicrofluidic device and its use in carrying out the synthesis andamplification of several DNA constructs of up to 1 kb in length. Thesynthesis reactions were carried out using polymerase construction andamplification (PCA) and the products amplified by PCR.

The microfluidic device was fabricated from PDMS and it was necessary touse a non-ionic surfactant, n-Dodecyl-β-D-maltoside (DDM) as apassivating agent.

Lee et al., Nucleic Acids Res. 2010; 38(8): 2514-21; Epub 2010 Feb. 21describes a microfluidic oligonucleotide synthesizer. A PDMS-basedmicrofluidic device was used as a miniaturized synthesizer forsolid-phase parallel synthesis of oligonucleotides.

SUMMARY OF THE INVENTION

The present inventors have devised a method for the combinatorialassembly of nucleic acid sequences which overcomes the disadvantages ofthe prior art and have now demonstrated that ligation of nucleic acidsequences to form a polynucleic acid sequence can be carried out on amicrofluidic device.

The method is a “one pot” method and is thus much quicker and moreconvenient to use than the hierarchical assembly approaches described inthe prior art which require the nucleic acid sequences to be assembledin a pre-defined order. In addition, the method does not require thecustom synthesis of nucleic acid sequences. The method makes use ofoligonucleotide linkers that can be attached to any standardized nucleicacid sequence, allowing for the quick and simple assembly of polynucleicacid sequences from multiple shorter nucleic acid sequences. In contrastto methods described in the prior art, the method of the presentinvention does not require a polymerase and does not involve thetechniques of PCR (polymerase chain reaction) or PCA (polymeraseconstruction and amplification or polymerase cycling assembly).

In the methods described in the prior art, the nucleic acid sequencesare typically prepared by PCR of a template with custom primers uniqueto each junction between parts, which requires the preparation ofnumerous primers. Moreover, PCR inherently is an error-prone operation,even with today's low error rate polymerases. Difficulties in handlingrepetitive sequences with fragments, or repeated copies of the samefragment also arise.

A very important feature of this technique is the ability to performthis pairing with no PCR reactions or novel oligo primers, using onlypre-designed and available DNA fragments, regardless of the pairing tobe performed. Thus, careful preparation of the required oligos andleft-right portions of each part allows unique combinations of parts tobe built with little delay, no additional oligo synthesis, and lowererror rates.

In a first aspect, the present invention provides a method for theassembly of a polynucleic acid sequence from a plurality of nucleic acidsequences in which the polynucleic acid sequence is of a formulaN_(n+1), in which N represents a nucleic acid sequence and where n is 1or greater than 1 and each N may be the same or a different nucleic acidsequence, in which the method comprises:

-   -   (i) providing a first nucleic acid sequence N1 which has an        oligonucleotide linker sequence L1^(3′) at the 3′-end of the        nucleic acid sequence;    -   (ii) providing a second nucleic acid sequence N2 which        optionally has an oligonucleotide linker sequence L2^(3′) at the        3′-end of the nucleic acid sequence and which has an        oligonucleotide linker sequence L2^(5′) at the 5′-end of the        nucleic acid sequence,        -   wherein the 5′-end linker sequence L2^(5′) of nucleic acid            sequence N2 is complementary to the 3′-end linker sequence            L1^(3′) of nucleic acid sequence N1,    -   (iii) optionally providing one or more additional nucleic acid        sequences N, wherein nucleic acid sequence N2 has an        oligonucleotide linker sequence L2^(3′) at the 3′-end of the        nucleic acid sequence, and wherein said one or more additional        nucleic acid sequences N comprises a terminal additional nucleic        acid sequence NZ, and wherein each additional nucleic acid        sequence N has an oligonucleotide linker sequence at its 3′-end,        wherein said terminal additional nucleic acid sequence NZ        optionally lacks an oligonucleotide linker sequence at its        3′-end and wherein each additional nucleic acid sequence N has        an oligonucleotide linker sequence at its 5′-end,        -   wherein for the first additional nucleic acid sequence N3            the 5′-end linker sequence L3^(5′) is complementary to the            3′-end linker sequence L2^(3′) of nucleic acid sequence N2            and for each second and subsequent additional nucleic acid            sequence N the 5′-end linker sequence is complementary to            the 3′-end linker sequence of the respective preceding            additional nucleic acid sequence;        -   and    -   (iv) ligating said nucleic acid sequences to form said        polynucleic acid sequence;        wherein at least step (iv) is carried out on a microfluidic        device.

DEFINITIONS

As used herein, “microfluidic device” means a device for manipulatingminute amounts of fluid, usually microliters (μL or μl) or nanoliters(nL or nl). Such devices are known in the art. They are typicallysubstantially planar and frequently contain features such as chambers,channels and/or valves. Their dimensions are typically in the region ofa few cm by a few cm. Typically, when a microfluidic device contains aplurality of chambers, they are linked to each other via fluid channelswhich can contain valves. Microfluidic devices can be fabricated from avariety of materials, such as glass and polydimethylsiloxane (PDMS). Theterms “microfluidic device”, “microfluidic chip” and “microfluidicplatform” are used interchangeably.

As used herein, when a method or a step of a method is described asbeing carried out “on chip” or “on-chip”, this means that the method ora step of a method is carried out on a microfluidic device. Conversely,when a method or step of a method is described as being carried out “offchip” or “off-chip”, this means that the method or step of a method isnot carried out on a microfluidic device, in other words the method orstep of a method is carried out away from or separately from amicrofluidic device. For example, aspects of the method claimed hereincan be carried out off-chip, typically using tubes and pipettes.

As used herein, the term “polynucleic acid sequence” means a polymer ofnucleic acids.

As used herein, the term “nucleic acid” means a polymer of nucleotides.Nucleotides are sometimes referred to as bases (in single strandednucleic acid molecules) or as base pairs (bp, in double stranded nucleicacid molecules). The term “nucleic acid” is used interchangeably hereinwith the term “part” and with the term “polynucleotide”. A “nucleicacid” or “polynucleotide” as defined herein includes a plurality ofoligonucleotides as defined herein.

Nucleic acids for use in the present invention are typically thenaturally-occurring nucleic acids DNA or RNA, but can also be artificialnucleic acids such as PNA (peptide nucleic acid), LNA (locked nucleicacid), UNA (unlocked nucleic acid), GNA (glycol nucleic acid) and TNA(threose nucleic acid). Nucleic acids such as DNA for use in theinvention can be synthetic or natural.

Nucleic acids for use in the present invention typically consist of thenucleotides adenine (A), cytosine (C), guanine (G), thymine (T) anduracil (U). Modified nucleotides that can also be used in the presentinvention include 4-acetylcytidine, 5-(carboxyhydroxylmethyl)uridine,2-O-methylcytidine, 5-carboxymethylaminomethyl-2-thiouridine,5-carboxymethylamino-methyluridine, dihydrouridine,2-O-methylpseudouridine, 2-O-methylguanosine, inosine,N6-isopentyladenosine, 1-methyladenosine, 1-methylpseudouridine,1-methylguanosine, 1-methylinosine, 2,2-dimethylguanosine,2-methyladenosine, 2-methylguanosine, 3-methylcytidine,5-methylcytidine, N6-methyladenosine, 7-methylguanosine,5-methylaminomethyluridine, 5-methoxyaminomethyl-2-thiouridine,5-methoxyuridine, 5-methoxycarbonylmethyl-2-thiouridine,5-methoxycarbonylmethyluridine, 2-methylthio-N6-isopentenyladenosine,uridine-5-oxyacetic acid-methylester, uridine-5-oxyacetic acid,wybutoxosine, wybutosine, pseudouridine, queuosine, 2-thiocytidine,5-methyl-2-thiouridine, 2-thiouridine, 4-thiouridine, 5-methyluridine,2-O-methyl-5-methyluridine and 2-O-methyluridine.

The length of a nucleic acid sequence or polynucleotide can be measuredin terms of the number of nucleotides it contains. The term “kilobase”(kb) means 1000 nucleotides.

As used herein, the term “oligonucleotide” means a polymer ofnucleotides (i.e. at least 2 nucleotides) that is shorter in length thana “nucleic acid” as defined herein. The term “oligonucleotide” issometimes abbreviated herein to “oligo”. Typically, an oligonucleotideconsists of up to 40 nucleotides or bases, more typically up to 60nucleotides or bases. Typically, an oligonucleotide is sufficientlyshort that it has no secondary or tertiary structure.

As used herein, the terms “3′” (“3 prime”) and “5′” (“5 prime”) taketheir usual meanings in the art, i.e. to distinguish the ends of anucleic acid molecules. As used herein, the terms 3′ and 5′ are alsoreferred to using the nomenclature 5′ and 3′. Nucleic molecules eachhave a 5′ and a 3′ end. Nucleic acids are synthesised in vivo in a 5′ to3′ direction, and nucleic acid sequences are conventionally written in a5′ to 3′ direction.

As used herein, the term “digestion” means cutting out. Typically,digestion is carried out using a restriction enzyme. For example, arestriction enzyme can be used to cut out or digest a nucleic acidsequence from a vector.

As used herein, the term “ligating” means joining together.

As used herein, the term “overhang” means a stretch of unpairednucleotides at the end of a nucleic acid, polynucleotide oroligonucleotide.

As used herein, the term “synthetic genome” means a polynucleic acidsequence that contains the information for a functioning organism ororganelle to survive and, optionally, replicate itself. The genome canbe completely or partially constructed from components that have beenchemically synthesized (e.g. synthetic DNA) or from copies of chemicallysynthesized nucleic acid components. The synthetic genome can be acompletely synthetic genome, i.e. constructed entirely from nucleic acidthat has been chemically synthesized, or from copies of chemicallysynthesized nucleic acid components. The synthetic genome canalternatively be a partially synthetic genome, i.e. constructedpartially from nucleic acid that has been chemically synthesized andpartially from naturally occurring nucleic acid. A partially syntheticgenome as defined herein can include nucleic acid derived from anyspecies of prokaryote or eukaryote, and/or elements from differentspecies.

In all definitions, the singular and plural are used interchangeably.

DETAILED DESCRIPTION OF THE INVENTION

The present invention provides a method for the assembly of apolynucleic acid sequence from a plurality of nucleic acid sequencesthat is at least partially carried out on a microfluidic device. Themethod of the invention thus involves the production of a long nucleicacid sequence from a number of shorter nucleic acid sequences.

The method of the present invention is described in detail inInternational Patent Application No. PCT/GB2009/002917 (WO 2010/070295).

In the method of the invention, the nucleic acid sequences are assembledsuch that the polynucleic acid includes a plurality of nucleic acidsequences in a predetermined order. For example, the method of theinvention can be used to assemble a gene or series of genes togetherwith their associated regulatory and control elements, thus producing acomplete operon. The method of the invention is thus useful in thecombinatorial assembly of genetic pathways, for example metabolicpathways and synthetic pathways. In one embodiment, the method of theinvention is useful in the production of a synthetic genome. Forexample, the method of the invention is useful for the production of asynthetic genome in a host cell such as a bacterial cell.

In the method of the present invention, the polynucleic acid sequence isof a formula N_(n+1), in which N represents a nucleic acid sequence andwhere n is 1 or greater than 1. The method of the present invention istherefore used to produce a polynucleic acid sequence containing 2 ormore nucleic acid sequences. The polynucleic acid sequence produced bythe method of the present invention is not limited to a polynucleic acidsequence having a particular number of nucleic acid sequences or parts.However, the polynucleic acid sequence typically contains from 2 to 60,typically from 2 to 50, typically from 2 to 40, typically from 2 to 35,typically from 2 to 30, typically from 2 to 20, typically from 3 to 15,from 4 to 10, from 5 to 9, from 6 to 8 nucleic acid sequences.Typically, the polynucleic acid sequence contains 2, 3, 4, 5, 6, 7, 8,9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30, 35, 40, 45, 48,50, 52, 54, 56, 58 or 60 nucleic acid sequences. Thus, n is typically 2,3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19, 20, 25, 30,35, 40, 45, 48, 50, 52, 54, 56, 58 or 60 and the polynucleic acidsequence produced is typically of the formula N₂ to N₁₀, i.e. thepolynucleic acid sequence produced is of the formula N₂, N₃, N₄, N₅, N₆,N₇, N₈, N₉ or N₁₀, or alternatively the polynucleic acid sequenceproduced is of the formula N₁₁, N₁₂, N₁₃, N₁₄, N₁₅, N₁₆, N₁₇, N₁₈, N₁₉,N₂₀, N₂₅, N₃₀, N₃₅, N₄₀, N₄₅, N₄₈, N₅₀, N₅₂, N₅₄, N₅₆, N₅₈ or N₆₀.

The polynucleic acid produced by the method of the present inventiontypically comprises at least 1000 nucleotides. Typically, thepolynucleic acid produced by the method of the present inventioncomprises between 1000 and 50000 nucleotides, typically between 2000 and40000 nucleotides, typically between 3000 and 30000 nucleotides,typically between 4000 and 25000 nucleotides, typically between 5000 and20000 nucleotides, typically between 6000 and 15000 nucleotides,typically between 7000 and 13000 nucleotides, typically between 8000 and12000 nucleotides, typically between 9000 and 11000 nucleotides,typically around 10000 nucleotides. Typically, the polynucleic acidsequence is from 10 kb to 30 kb. However, the polynucleic acid can befrom 10 kb to 30 kb, 40 kb, 50 kb, 75 kb, 100 kb, 120 kb, 140 kb or 150kb. In some embodiments, the polynucleic acid is at least 150 kb, atleast 200 kb, at least 250 kb, at least 300 kb, at least 350 kb, atleast 400 kb, at least 450 kb or at least 500 kb in length.

The method of the invention can be used to obtain a plurality ofpolynucleic acid sequences. For example, the method of the invention canbe carried out multiple times in parallel, with the result being aplurality of polynucleic acid sequences. The polynucleic acid sequencesobtained by carrying out the method of the invention multiple times canthen be joined together into a longer polynucleic acid sequence, forexample using the method of the present invention but where N representsa polynucleic acid sequence rather than a nucleic acid sequence. Thismethod can therefore be used to produce longer polynucleic acidsequences.

In the method of the invention, each N may be the same or a differentnucleic acid sequence. The method of the invention can therefore be usedfor the combinatorial assembly of the same or different nucleic acidsequences. Typically, each N is a different nucleic acid sequence.However, the method of the invention can also be used to produce apolynucleic acid sequence comprising a number of nucleic acids which arethe same, for example to increase the copy number of a protein-codingsequence. This embodiment of the invention is useful in the preparationof a combinatorial library of nucleic acid sequences as describedherein.

The nucleic acid sequences used in the present invention can be codingor non-coding sequences. Typically, each nucleic acid sequence used inthe present invention is a protein coding sequence or a regulatory orcontrol element.

The nucleic acid sequences used in the present invention can be obtainedfrom any suitable source. For example, the nucleic acid sequences can besynthesised for use in the invention or can be obtained from a naturalsource. Conveniently, the nucleic acid sequences used in the presentinvention can be sourced from the BioBricks™ registry of standard parts(Cambridge, Mass.; partsregistry.org). BioBricks™ parts are nucleicacids of defined structure and function. The method of the presentinvention can therefore be used to assemble existing BioBricks™ partsavailable from the BioBricks™ registry of standard parts.

Protein coding sequences for use in the present invention includesequences that encode proteins that are part of metabolic or othergenetic pathways. Protein coding sequences for use in the presentinvention also include sequences that encode experimentally usefulproteins, such as reporter proteins. Suitable reporter proteins for usein the present invention include coloured proteins such as lacZa,fluorescent proteins such as RFP or GFP, and proteins that conferantibiotic resistance. Reporter genes are linked to a test promoter,enabling activity of the promoter gene to be determined by detecting thepresence of the reporter gene product.

For example, the method of the invention can be used to produce a DNAassembly in which a ribosome binding site (RBS) is inserted between atranscriptional promoter and a reporter protein, for example lacZa ormRFP1. Proper insertion of this RBS results in a complete operon forexpression of the reporter protein, resulting in a phenotypic change.

Regulatory or control elements for use in the present invention includepromoters, operators, repressors, ribosome-binding sites, internalribosome entry sites (IRESs) origins of replication, enhancers,polyadenylation regions, splice donor and acceptor sites,transcriptional termination sequences, 3′ UTRs and 5′ UTRs.

Promoters are regions of DNA that facilitate the transcription of aparticular gene by including a binding site for RNA polymerase.Promoters typically lie upstream of the gene whose transcription theycontrol. Promoters for use in the invention include constitutive andinducible promoters.

In a first step (i), the method of the present invention comprisesproviding a first nucleic acid sequence N1 which has an oligonucleotidelinker sequence L1^(3′) at the 3′-end of the nucleic acid sequence.

In one embodiment of the invention, the first nucleic acid sequence N1also has an oligonucleotide linker sequence L1^(5′) at the 5′-end of thenucleic acid sequence.

In a second step (ii), the method of the present invention comprisesproviding a second nucleic acid sequence N2 which optionally has anoligonucleotide linker sequence L2^(3′) at the 3′-end of the nucleicacid sequence and which has an oligonucleotide linker sequence L2^(5′)at the 5′-end of the nucleic acid sequence, wherein the 5′-end linkersequence L2^(5′) of nucleic acid sequence N2 is complementary to the3′-end linker sequence L1^(3′) of nucleic acid sequence N1. The secondnucleic acid sequence N2 therefore has an oligonucleotide linkersequence L2^(5′) at its 5′-end, and optionally also has anoligonucleotide linker sequence L2^(3′) at the 3′-end of the nucleicacid sequence.

In one embodiment of the invention, the second nucleic acid sequence N2also has an oligonucleotide linker sequence L2^(3′) at the 3′-end of thenucleic acid sequence. For example, the second nucleic acid sequence N2has an oligonucleotide linker sequence L2^(3′) at the 3′-end of thenucleic acid sequence in the embodiment where the polynucleic acidsequence is of the formula N_(≧3), i.e. wherein the polynucleic acidsequence is comprised of 3 or more nucleic acid sequences N.

The third step (iii) of the method of the present invention is optional.The third step (iii) of the method of the invention is present in theembodiment where the polynucleic acid sequence is of the formula N_(≧3),i.e. wherein the polynucleic acid sequence is comprised of 3 or morenucleic acid sequences N.

Thus in one embodiment of the invention, in which step (iii) of themethod of the invention is not present, the method of the presentinvention comprises:

-   -   (a) providing a first nucleic acid sequence N1 which has an        oligonucleotide linker sequence L1^(3′) at the 3′-end of the        nucleic acid sequence;    -   (b) providing a second nucleic acid sequence N2 which has an        oligonucleotide linker sequence L2^(5′) at the 5′-end of the        nucleic acid sequence,        -   wherein the 5′-end linker sequence L2^(5′) of nucleic acid            sequence N2 is complementary to the 3′-end linker sequence            L1^(3′) of nucleic acid sequence N1;        -   and    -   (c) ligating said nucleic acid sequences to form said        polynucleic acid sequence;        wherein at least step (c) is carried out on a microfluidic        device.

In this embodiment of the invention, the polynucleic acid sequence is ofthe formula N₂, i.e. the polynucleic acid sequence is comprised of 2nucleic acid sequences, N1 and N2.

In some embodiments, the first nucleic acid sequence N1 also has anoligonucleotide linker sequence L1^(5′) at the 5′-end of the nucleicacid sequence. In some embodiments, the second nucleic acid sequence N2also has an oligonucleotide linker sequence L2^(3′) at the 3′-end of thenucleic acid sequence. In some embodiments, the 5′-end linker sequenceL1^(5′) of nucleic acid sequence N1 is complementary to the 3′-endlinker sequence L2^(3′) of nucleic acid sequence N2. In this embodimentof the invention, the nucleic acid is circular, and is typicallycircular DNA.

In the optional third step (iii), the method of the present inventioncomprises providing one or more additional nucleic acid sequences N,wherein nucleic acid sequence N2 has an oligonucleotide linker sequenceL2^(3′) at the 3′-end of the nucleic acid sequence, and wherein said oneor more additional nucleic acid sequences N comprises a terminaladditional nucleic acid sequence NZ, and wherein each additional nucleicacid sequence N has an oligonucleotide linker sequence at its 3′-end,wherein said terminal additional nucleic acid sequence NZ optionallylacks an oligonucleotide linker sequence at its 3′-end and wherein eachadditional nucleic acid sequence N has an oligonucleotide linkersequence at its 5′-end, wherein for the first additional nucleic acidsequence N3 the 5′-end linker sequence L3^(5′) is complementary to the3′-end linker sequence L2^(3′) of nucleic acid sequence N2 and for eachsecond and subsequent additional nucleic acid sequence N the 5′-endlinker sequence is complementary to the 3′-end linker sequence of therespective preceding additional nucleic acid sequence.

In step (iii), the 5′-end linker sequence of each second and subsequentadditional nucleic acid sequence N is complementary to the 3′-end linkersequence of the respective preceding additional nucleic acid sequence.In other words, Li^(5′) is complementary to L(i−1)^(3′).

In some embodiments, the first nucleic acid sequence N1 also has anoligonucleotide linker sequence L1^(5′) at the 5′-end of the nucleicacid sequence. In some embodiments, the terminal additional nucleic acidsequence NZ also has an oligonucleotide linker sequence LZ^(3′) at the3′-end of the nucleic acid sequence. In some embodiments, the 5′-endlinker sequence L1^(5′) of nucleic acid sequence N1 is complementary tothe 3′-end linker sequence LZ^(3′) of nucleic acid sequence NZ. In thisembodiment of the invention, the nucleic acid is circular, and istypically circular DNA.

In one embodiment of the invention, the method of the present inventioncomprises:

-   -   (i) providing a first nucleic acid sequence N1 which has an        oligonucleotide linker sequence L1^(3′) at the 3′-end of the        nucleic acid sequence;    -   (ii) providing a second nucleic acid sequence N2 which has an        oligonucleotide linker sequence L2^(3′) at the 3′-end of the        nucleic acid sequence and which has an oligonucleotide linker        sequence L2^(5′) at the 5′-end of the nucleic acid sequence,        -   wherein the 5′-end linker sequence L2^(5′) of nucleic acid            sequence N2 is complementary to the 3′-end linker sequence            L1^(3′) of nucleic acid sequence N1;    -   (iii) providing a third nucleic acid sequence N3 which has an        oligonucleotide linker sequence L3^(5′) at the 5′-end of the        nucleic acid sequence,        -   wherein the 5′-end linker sequence L3^(5′) of nucleic acid            sequence N3 is complementary to the 3′-end linker sequence            L2^(3′) of nucleic acid sequence N2;        -   and    -   (iv) ligating said nucleic acid sequences to form said        polynucleic acid sequence;        wherein at least step (iv) is carried out on a microfluidic        device.

In this embodiment of the invention, the polynucleic acid sequence is ofthe formula N₃, i.e. the polynucleic acid sequence is comprised of 3nucleic acid sequences, N1, N2 and N3.

In some embodiments, the first nucleic acid sequence N1 also has anoligonucleotide linker sequence L1^(5′) at the 5′-end of the nucleicacid sequence. In some embodiments, the third nucleic acid sequence N3also has an oligonucleotide linker sequence L3^(3′) at the 3′-end of thenucleic acid sequence. In some embodiments, the 5′-end linker sequenceL1^(5′) of nucleic acid sequence N1 is complementary to the 3′-endlinker sequence L3^(3′) of nucleic acid sequence N3. In this embodimentof the invention, the nucleic acid is circular, and is typicallycircular DNA.

In a fourth step (iv), the method of the present invention comprisesligating said nucleic acid sequences to form said polynucleic acidsequence. At least step (iv) of the method of the invention is carriedout on a microfluidic device.

Typically, step (iv) of the method of the invention is carried out usingDNA ligase. DNA ligase is an enzyme that links together two DNA strandsthat have a double-stranded break. Any type of commercially availableDNA ligase can be used in the present invention, for example T4 DNAligase available from New England Biolabs, MA. Any of the mammalian DNAligases (DNA ligase I, DNA ligase II, DNA ligase III, DNA ligase IV) canbe used in the present invention. Step (iv) of the method of theinvention can alternatively be carried out using RNA ligase.

In some embodiments, step (iv) of the method of the invention is carriedout without using a ligase. For example, in one embodiment, step (iv) ofthe method of the invention is carried out using chemical ligation. Anysuitable method for carrying out chemical ligation can be used, forexample using cyanogen bromide as a condensing agent or using hydrogenperoxide.

In one embodiment of the invention, the nucleic acid sequences Ntogether with their oligonucleotide linker sequences are purifiedimmediately prior to step (iv). Any suitable method can be used topurify the nucleic acid sequences. By “purifying the nucleic acidsequences” is meant removing any unbound oligonucleotide linkersequences (also referred to herein as part/linker oligos).

Purification can be carried out in a number of separate steps, forexample two separate steps as illustrated in FIGS. 9 and 10. In oneembodiment, a first purification step is carried out to remove unboundoligonucleotide linker sequences that bind to the 3′-end of a nucleicacid sequence and a second step is carried out to remove unboundoligonucleotide linker sequences that bind to the 5′-end of a nucleicacid sequence. In some embodiments, a number of different purificationsteps will be needed, typically one per oligonucleotide linker sequenceattached to the nucleic acid sequences to be assembled using the methodof the invention.

As set out above, at least step (iv) of the method of the invention iscarried out on a microfluidic device. The step of purifying the nucleicacid sequences immediately prior to step (iv) can either be carried outon-chip or off-chip (as defined herein).

Thus, in one embodiment, the nucleic acid sequences are purified on themicrofluidic device. In this embodiment, the nucleic acid sequences aretypically purified by biotin-based methods such as biotin-streptavidinpurification, for example biotin-streptavidin purification usingstreptavidin-coated beads, typically streptavidin-coated magnetic beads.Such beads are of a size suitable for use in a microfluidic device asdescribed herein, and so the diameter of such beads will be in the nm orμm range, for example from 500 nm to 5 μm, from 700 nm to 4 μm, from 800nm to 3 μm, from 900 nm to 2 μm or around 1 μm.

The nucleic acid sequences can be purified on the same microfluidicdevice on which step (iv) is carried out or alternatively thepurification step and the ligation step (iv) can be carried out onseparate microfluidic devices. In one embodiment, purification iscarried out using one or more microfluidic devices prior to ligationstep (iv) being carried out on a different microfluidic device.Purification of multiple nucleic acid sequences can be carried outsimultaneously either on one microfluidic device or on a plurality ofmicrofluidic devices, which may or may not be linked to the microfluidicdevice on which the ligation step is carried out.

In one embodiment, the nucleic acid sequences are purified bybiotin-streptavidin purification, for example as shown in FIG. 11. Thispurification approach targets a specific DNA sequence by annealing DNAto a biotinylated oligo. This purification approach utilizes abiotinylated oligo that is complementary to an overhang on thepart-linker pair or the pathway assembly. The oligo anneals to the DNAfragment and then the pair is washed over streptavidin-coated beads.These beads can be magnetic or otherwise easily purified from solution.This purification approach was used in Example 1 herein.

In an alternative embodiment, the nucleic acid sequences are purifiedoff-chip, as defined herein. In other words, purification does not takeplace on the microfluidic device. In this embodiment, the nucleic acidsequences are typically purified using DNA purification spin columns orgel extraction. For example, the part-linker pairs can be purified usingthe QiaQuick™ PCR purification kit (Qiagen™) or via gel electrophoresisand extraction via the QiaQuick™ gel extraction kit (Qiagen™). Gel-basedpurification was used in Example 4 herein. Many other suitable methodsof purifying nucleic acid sequences will be known to the skilled personand can be used in the present invention, for example high performanceliquid chromatography (HPLC) and nuclease treatment that selectivelydestroys unbound oligonucleotides.

Once the method of the invention has been carried out to produce thepolynucleic acid sequence, the polynucleic acid sequence can betransformed into suitable cells to verify that the assembly has beensuccessful. Suitable cells include chemically competent E. coli(available from Invitrogen, CA or New England Biolabs, MA). Thesuccessful assembly can then be verified, e.g. by plating out the cellsand counting coloured/colourless colonies, for example by counting redor green colonies as described in Example 1 herein.

In one embodiment of the invention, each of the nucleic acid sequences Nis provided with an overhang at one or both ends. In some embodiments,the nucleic acid sequences N have an overhang only at one end, eitherthe 3′-end or the 5′-end. Typically, each nucleic acid sequence N has anoverhang at both the 3′-end and the 5′-end. This embodiment of theinvention is illustrated in FIGS. 6 and 7.

Typically, the overhang at one or both ends of each nucleic acidsequence is produced by digestion with one or more restriction enzymes.For example, in some embodiments of the invention one or more of thenucleic acid sequences is stored in a vector prior to use in the methodof the invention, as shown in FIGS. 6 and 7. Typically, BioBricks™parts, i.e. nucleic acids from the BioBricks™ registry (Cambridge,Mass.), are stored in this fashion. In this embodiment, a restrictionenzyme is used to cut the nucleic acid sequence out from the vector inwhich it is stored before use in the method of the invention.

The overhang at one or both ends of the nucleic acid sequence can bepalindromic or non-palindromic.

Any restriction enzyme can be used in the present invention. Suitablerestriction enzymes for use in the invention include restriction enzymesthat produce single-stranded overhangs. Typical restriction enzymes foruse in the present invention are Type IIS restriction enzymes, whichcleave at sites away from their recognition site.

Suitable restriction enzymes for use in the invention include EcoRI,SpeI, SapI, EarI and PstI. The restriction enzyme is typically EarI.

In one embodiment, in which the nucleic acid sequence has an overhang atboth ends, the overhang at the 5′-end of the nucleic acid sequence canbe produced by digestion with EcoRI and the overhang at the 3′-end ofthe nucleic acid sequence can be produced by digestion with SpeI. Theoverhangs produced by EcoRI and SpeI are palindromic. EcoRI and SpeI aretypically used in this fashion to prepare BioBricks™ parts, i.e. nucleicacids from the BioBricks™ registry, and leave standard overhangs at the5′-end and the 3′-end of the nucleic acid sequence.

In another embodiment, the overhang at the 5′-end of the nucleic acidsequence or at the 3′-end of the nucleic acid sequence can be producedby digestion with SapI or EarI. The overhangs produced by SapI and EarIare non-palindromic.

The overhangs produced by EcoRI/SpeI and SapI/EarI are showndiagrammatically in FIG. 8.

The overhangs on each nucleic acid sequence N are typically produced bydigestion with the same restriction enzyme or combination of restrictionenzymes. Alternatively, the overhangs on different nucleic acidsequences N can be produced using different restriction enzymes orcombinations of restriction enzymes. For example, one nucleic acidsequence N can be designed to be cut by EcoRI/SpeI and another nucleicacid sequence N can be designed to be cut by EcoRI/PstI. However, ineither of these embodiments, there will typically be one standardoverhang on each of the nucleic acid sequences.

In one embodiment, the overhang is 3 or 4 nucleotides in length.However, the overhang can be of a different length, for example 2nucleotides or 5, 6, 7, 8, 9 or 10 nucleotides in length.

In one embodiment, the overhang at the 3′-end of the nucleic acidsequence is the same for each nucleic acid sequence. In anotherembodiment, the overhang at the 5′-end of the nucleic acid sequence isthe same for each nucleic acid sequence. In another embodiment, theoverhang at the 3′-end of the nucleic acid sequence and the overhang atthe 5′-end of the nucleic acid sequence is the same for each nucleicacid sequence. The overhang can therefore be the same or different ateach end of the nucleic acid sequence.

In the embodiment in which the overhang at the 3′-end of the nucleicacid sequence or at the 5′-end of the nucleic acid sequence is the samefor each nucleic acid sequence, the nucleic acid sequences can bedesigned such that the same overhang is produced after restrictiondigest, i.e. after digestion with a restriction enzyme.

In one embodiment, the present invention encompasses a library ofnucleic acid sequences with appropriate overhangs ready for use in theinvention.

Each nucleic acid sequence used in the method of the present inventionhas an oligonucleotide linker sequence at the 3′-end, at the 5′-end orat both the 3′-end and the 5′-end of the nucleic acid sequence, asdescribed herein.

In one embodiment, the present invention encompasses a library ofnucleic acid sequences together with appropriate oligonucleotide linkersequences ready for use in the invention.

The oligonucleotide linker sequences used in the present invention aretypically double stranded. Typically, the oligonucleotide linkersequences are partially double stranded. That is to say, each of the3′-end linker sequences and each of the 5′-end linker sequences used inthe method of the present invention is typically partially doublestranded. By “partially double stranded” is meant that either the 3′-endor the 5′-end of the linker sequence or both has an overhang. In thisembodiment, each of the two strands of the 3′-end linker sequences andthe 5′-end linker sequences have different numbers of nucleotides. Theresult of this is that each of the linker sequences has an overhang. Thelinker sequences can therefore be considered as being comprised of twoseparate single stranded oligonucleotide sequences of different lengths,with the result being that the linker sequences are partially doublestranded and have one or more overhangs. This embodiment of theinvention is illustrated in FIGS. 6 and 7.

In some embodiments of the invention, the overhang at one end of thenucleic acid sequence is complementary to the overhang on the 3′-endlinker sequence and/or to the overhang on the 5′-end linker sequence.This embodiment of the invention is also illustrated in FIGS. 6 and 7.In these embodiments of the invention, each of the nucleic acidsequences used in the method of the invention is attached to its said3′-end linker sequence and to its said 5′-end linker sequence byoligonucleotide annealing and ligation. In some embodiments, where thenucleic acid is DNA, ligation is carried out using DNA ligase.

Typically, one or more of the nucleic acid sequences is attached to its3′-end linker sequence and/or to its 5′-end linker sequence by ligation.More typically, each of the nucleic acid sequences is attached to its3′-end linker sequence and to its 5′-end linker sequence by ligation.Typically, ligation is carried out using a ligase, typically DNA ligase.

In some embodiments, the oligonucleotide linker sequences used in thepresent invention are single stranded.

In some embodiments, the nucleic acid sequences are provided in a vectorand are cut out or digested from the vector using a restriction enzyme.

Accordingly, in one embodiment, the present invention provides a methodfor the assembly of a polynucleic acid sequence from a plurality ofnucleic acid sequences in which the polynucleic acid sequence is of aformula N_(n+1), in which N represents a nucleic acid sequence and wheren is 1 or greater than 1 and each N may be the same or a differentnucleic acid sequence, in which the method comprises:

-   -   (i) providing a first nucleic acid sequence N1 in a vector,        using a restriction enzyme to cut the nucleic acid sequence out        from the vector and ligating an oligonucleotide linker sequence        L1^(3′) to the 3′-end of the nucleic acid sequence;    -   (ii) providing a second nucleic acid sequence N2 in a vector,        using a restriction enzyme to cut the nucleic acid sequence out        from the vector, optionally ligating an oligonucleotide linker        sequence L2^(3′) to the 3′-end of the nucleic acid sequence and        ligating an oligonucleotide linker sequence L2^(5′) to the        5′-end of the nucleic acid sequence,        -   wherein the 5′-end linker sequence L2^(5′) of nucleic acid            sequence N2 is complementary to the 3′-end linker sequence            L1^(3′) of nucleic acid sequence N1;    -   (iii) optionally providing one or more additional nucleic acid        sequence(s) N in one or more vector(s), using a restriction        enzyme to cut the nucleic acid sequence(s) out from the        vector(s), wherein nucleic acid sequence N2 has an        oligonucleotide linker sequence L2^(3′) at the 3′-end of the        nucleic acid sequence, and wherein said one or more additional        nucleic acid sequences N comprises a terminal additional nucleic        acid sequence NZ, and ligating an oligonucleotide linker        sequence to the 3′-end of each additional nucleic acid sequence        N, wherein said terminal additional nucleic acid sequence NZ        optionally lacks an oligonucleotide linker sequence at its        3′-end, and ligating an oligonucleotide linker sequence to the        5′-end of each additional nucleic acid sequence N,        -   wherein for the first additional nucleic acid sequence N3            the 5′-end linker sequence L3^(5′) is complementary to the            3′-end linker sequence L2^(3′) of nucleic acid sequence N2            and for each second and subsequent additional nucleic acid            sequence N the 5′-end linker sequence is complementary to            the 3′-end linker sequence of the respective preceding            additional nucleic acid sequence; and    -   (iv) ligating said nucleic acid sequences to form said        polynucleic acid sequence;        wherein at least step (iv) is carried out on a microfluidic        device.

In one embodiment, the digestion and ligation steps are carried out intwo separate reactions. Alternatively, the digestion and ligation stepscan be combined in a single reaction.

Typically, when the digestion and ligation steps are combined, thereaction is carried out by alternating between the digestion and theligation step. This is typically done by alternating betweentemperatures suitable for digestion and ligation, typically multipletimes. For example, the reaction can be carried out by alternating atleast 1, 2, 3, 4, 5 or 6 times between a temperature suitable fordigestion and a temperature suitable for ligation. For example, thereaction can be carried out under conditions in which the temperature isalternated 2, 3, 4, 5 or 6 times between a temperature expected to beoptimal for digest activity (for example from 30° C. to 40° C.,typically from 33° C. to 39° C., more typically from 37° C. to 38° C.,more typically 37° C.) and a temperature expected to be near optimal forligation activity (for example from 12° C. to 19° C., typically from 13°C. to 18° C., more typically from 15° C. to 17° C., more typically 16°C.). This embodiment of the invention is typically carried out using athermocycle. This embodiment is illustrated in Example 4 herein and inFIG. 44.

This embodiment of the invention has a number of advantages, includingmaking downstream purification easier, saving on oligo costs, andshortening the assembly process. Combining the digestion and ligationreactions means that much less of the ligation oligos needs to be used,as any incorrect re-ligations will be re-digested by the restrictionenzyme in the next cycle. This makes the downstream purification easieras there are fewer contaminating oligos. Running a single reactionsimplifies the entire process by reducing a step and also increases thepurification efficiency.

One embodiment of the present invention is as follows.

The approach described here enables the assembly of many differentpathways from a small collection of standard parts by assemblingmultiple parts in a single reaction step. Unlike similar “one pot”assembly approaches conducted previously, this approach requires neithersynthesis of custom oligos for assembly nor that parts be assembled in apre-defined order. By standardizing parts, it is possible to expendeffort once to prepare components and then reuse them to generate manypathways in a rapid, highly parallelizable reaction.

This assembly process involves three phases: part preparation,part-linker fusion, and pathway assembly (see FIG. 1).

The part preparation is outside of the assembly cycle. Extra work is putinto the design and preparation of parts in order to reduce the timerequired for assembly (see FIG. 2).

All parts can be stored in plasmids such that they can be cut out in astandard form. The offset cutter SapI is used as an example through thisdescription, but this approach is by no means limited by the particularenzyme. SapI recognizes a 7 bp sequence and leaves a 3 bp overhang thatcan be anything. Parts are designed to be cut out by digestion withSapI, leaving 3 bp overhangs on both ends. The 3 bp overhang on the3′-end of the part is defined to be a standard 3 bp sequence common forall parts in this format. The 3 bp overhang on the 5′-end of the part isdefined to be something other than this standard 3 bp sequence. The partsequences are all defined to not contain any extra recognition sites forSapI.

Parts are split into two regions: a short beginning region and the restof the part. The beginning region (approximately 10 bp) is defined byoligos. The remainder of the part is stored in a plasmid as describedabove. When the plasmid is cut, the truncated part is released. Uponligation with the oligos, the part is reconstituted except for anoverhang on the front (e.g. 10 bp).

The process for preparing part A with defined overhangs involves:

-   -   1. Cutting the plasmid prep with SapI    -   2. Ligate oligos 1_(A) and 2_(A) (both 5′-phosphorylated) to the        cut part    -   3. Also, add biotinylated oligo 5_(A) which binds to the        ligation product via non-covalent base pairing (e.g. 10 bp)    -   4. The complex is purified via the biotin (e.g. using magnetic        streptavidin beads)    -   5. The purified, ligated product is released by heating to break        the pairing with the biotin oligo    -   6. Oligos 3_(A) and 4_(A) are synthesized during the        construction of part A and stored in annealed form for use        during the assembly

Note that there may be other ways of obtaining the same final DNAstructure, such as de novo DNA synthesis or PCR methods (e.g. the NewEngland Biolabs USER™ (Uracil-Specific Excision Reagent) system). Theprepared parts that are output and stored from this phase include thecomplete part except for a long overhang (e.g. 10 bp) on one side and a3 bp overhang on the other side. The short 3 bp overhang is a standardsequence and the long overhang sequence is from the part.

Designing parts in this format needs to be done once. Afterstandardization, parts can then be reused in as many differentassemblies as desired. Prepared parts are stored, along with the helperoligos, and serve as input to the assembly phases. The same part oligoscan be used for all assemblies using the part. Purification and qualitycontrol during part preparation and storage ensures that the inputs tothe assembly process are of the highest quality.

Note that the above description assumes that the 3 bp overhang at theend of the part is standardized. A substantially identical process ispossible by standardizing the 3 bp overhang at the beginning of a partand defining oligos to complete the end of a part. The assembly processin either case is similar.

The long overhang after part preparation can be designed to form eithera 5′- or 3′-overhang, unlike many other assembly methods. In addition,it is possible to mix and match both types of overhangs in an assemblywithout needing to know beforehand which parts will be assembled. Asmart part design process would use different types of overhangs tomaximize the number of parts that can be assembled in one pot.

Also described herein is the computer aided design (CAD) tools used todesign the parts. The design of a part starts with the initial DNAsequence for a part, call it part A. This sequence is required to notinclude the recognition sequence for SapI (or any other enzyme used), ineither orientation. If required, the CAD system designs oligos and amutation strategy, such as the Quikchange mutagenesis (Stratagene) toremove these sites. This process takes into account the properties anduse of the sequence, preferring silent mutations in coding regions, forexample.

A (potentially modified) sequence for the complete part A is thenavailable. In a second step, this sequence is analyzed to locate anappropriate place to truncate part A into two parts: (1) the left mostpart, which will be created by the reverse complement of oligo 2_(A),(2) a truncated portion of part A, which consists of the remainder ofpart A. There are several considerations used by the program to locatethe position of this split. (a) The three base overhang created at theleft end of the truncated part A must satisfy several requirements. Itmust not be identical to the standard overhang forming the three basescar at the right end of every part. It must have an appropriate meltingtemperature, such that the ligation of annealed oligos 1_(A) and 2_(A)can be carried out efficiently. Typically, this would require a minimalGC content in the overhang. The length and melting temperature of oligo1_(A) must be sufficient to stabilize the 1_(A) and 2_(A) annealeddouble strand together at the ligation temperature. The overlap of oligo5_(A) with oligo 2_(A) must be sufficiently long and have a high enoughmelting temperature to stabilize the double stranded structure formed atthe ligation temperature. This overlap must be sufficiently short andhave low enough melting temperature to be disassociable during biotinpurification of part A. The output of the algorithm is then the locationof the split between the truncated part A and oligo 2_(A). This splitlocation also determines the three base overhang at the left end of partA and the length of oligo 2_(A). The second output of the algorithm isthe length of oligo 1_(A), which also determines the length of oligo5_(A). Oligos 3_(A) and 4_(A) are then easily defined using thesequences of oligos 5_(A) and 2_(A).

The lengths of oligos 1_(A), 2_(A), and 5_(A) are determined usingstandard search techniques within the sequence A. It is possible thatthis search will fail to yield good sequences, and may require redesignof the part to satisfy the assembly process, but this is a rare event.

Another output of the algorithm is the pair of PCR primers necessary toamplify the truncated part A, including the SapI cut sites at both ends,and the three by unique left overhang, and the standard three byoverhang on the right end.

In the part-linker fusion phase, parts are processed into assembly-readyparts (FIG. 3). Although assembly-ready parts depend on the desiredassembly, assembly-ready parts can likely be reused in many pathwayassemblies. The reactions in this phase are highly parallel and can bedone in constant time (e.g. the time to assemble the pathway isindependent of the number of parts contained in the pathway).

One reaction is done for every part junction that is desired.Biotin-based purification is described here, but other means ofpurification are of course also possible. For example, if part A andpart B need to be assembled in some pathway, then the following processis performed:

-   -   1. Ligate to the prepared part A the annealed oligos 3_(B) and        4_(B) that were constructed during the part preparation phase of        part B    -   2. The oligos for B form the standard 3 bp overhang and thus can        ligate with A easily    -   3. 4_(B) is designed to not ligate with part A. For example,        4_(B) may have a 3′-dideoxy nucleotide or may be “missing” a        base at the 3′-end    -   4. For purifying the correctly ligated product, 4_(B) is        biotinylated    -   5. The complex is purified via the biotin (e.g. using magnetic        streptavidin beads)    -   6. The purified, ligated product is released by heating to break        the pairing with the biotin oligo

The above process will create a molecule that contains the entirety ofpart A, followed by a 3 nt standard “scar” sequence, and then followedby the initial sequence of part B. We will refer to this fusion moleculeas A_(B). Both ends of this molecule will contain long overhangsmatching the beginning of part A and the beginning of part B. One canimagine a scarless version of this fusion process. For example, if the 3nt overhang were chewed back (e.g. via a nuclease) and a blunt ligationdone, then the scar would disappear. We will assume for simplicity inthis description that a 3 nt scar will appear between assembled parts.

The final phase is the assembly of the complete desired pathways usingin vitro ligation. The process for this phase is extremely simple (FIGS.4 and 5). As the overhangs should all match perfectly, only ligase isrequired in this reaction. Assume that parts A, B, Y, and Z are beingassembled to form a linear DNA. Suppose parts A and Z are standard partsadded at the beginning and end of all assemblies, and thus notnecessarily a part of the desired pathway.

-   -   1. All of the part-linker fusions of A_(B), B_(C), . . . , Y_(Z)        generated from the previous step are mixed together    -   2. DNA ligase is added    -   3. Add the biotinylated oligo 5_(A) that base pairs to the front        overhang of A    -   4. Pull out the biotin using magnetic streptavidin beads        effectively purifying away anything that does not begin with        part A    -   5. Purify away the biotinylated oligo 5_(A) by heating to break        the base pairing    -   6. Add the biotinylated oligo 4_(Z) which base pairs to the        overhang of Y    -   7. Pull out the biotin using magnetic streptavidin beads        effectively purifying away anything that does not end with part        Y    -   8. Purify away the biotinylated oligo 4_(A) by heating to break        the base pairing    -   9. The biotinylated oligo is purified away by heating to break        the base pairing

The resulting purified fragment will contain the assembly of parts A . .. Y_(Z). Complete dsDNA is present for the desired assembly of parts B .. . Y. Extra overhangs are present on the ends which can be blunted witha nuclease if so desired. If a circular DNA is desired, one can add afinal ligation step with Z_(A) which will complete the circle. Forexample, this circular DNA can then be cloned into bacteria forpropagation.

The time required for the pathway assembly phase depends on the natureof the pathways to be assembled. For a subset of the parts (i.e. partsthat don't have matching overhang sequences), this phase can be done inconstant time, independent of the size of the pathways to be assembled.However, if some parts have matching overhangs (e.g. if a part is usedmultiple times in the assembly), this assembly process needs to bebroken into multiple cycles such that the offending parts are assembledin different reactions before being combined.

The output of the assembly process is the purified, assembled pathway.Ultimately, sequencing will not be necessary due to the stringentquality control used during part preparation and the purification duringthe process. The purifications are described using biotin/streptavidinpurification steps. However, other methods such as length-based (e.g.gel electrophoresis) can also be substituted.

It is possible for an assembled pathway to be used as a new part viaidempotent assembly. If one wishes to use the assembly process toproduce a plasmid that can itself be used as a part (i.e. contains thecorrect placement of SapI sites), a couple of small changes need to bemade. During the part preparation phase for the first part (A), adifferent set of oligos that add back a SapI site instead ofreconstituting part A should be used. The last part during the assembly(Z) should also contain a SapI site in the proper location. All otheraspects of the assembly can remain the same.

In one embodiment, the present invention provides a method for theassembly of a polynucleic acid sequence as shown in FIG. 6.

In the part cloning phase, a part (in this case Part A) is prepared bycarrying out PCR using a forward primer (A_(F)) and a reverse primer(A_(R)). The part is designed to be in a standard form, with a standardoverhang at the 3′-end (S_(L)) and a standard overhang at the 5′-end(S_(P)), each of which is designed to be recognised by a particularrestriction enzyme. Conveniently, the part can be cloned into a plasmidfor storage before carrying out the part preparation phase.

In the part preparation phase, the plasmid containing the part isdigested with one or more restriction enzymes that recognises and cutsthe part at a predefined sequence, leaving standard overhangs at the3′-end (S_(L)) and at the 5′-end (S_(P)) of the part.

In the part/linker assembly phase, the part (in this case Part A) isligated and purified using one set of standard part oligos. These oligosbind at the 5′-end of the part. The standard part oligos include apartially double stranded linker oligonucleotide that consists of ashorter oligonucleotide (X¹ _(P2)) that binds to the overhang S_(P) atthe 5′-end of the part and a longer oligonucleotide (X¹ _(P1)) thatbinds directly to the 5′-end of the part on the strand which does nothave the overhang. X¹ _(P2) is complementary to X¹ _(P1) and since X¹_(P1) is longer than X¹ _(P2) a new overhang is created at the 5′-end ofthe part.

The standard part oligos also include a part purification oligo (X¹_(PP)) that binds to X¹ _(P2) and is partially complementary to X¹ _(P1)in the new region of overhang created when X¹ _(P2) binds to X¹ _(P1).The part purification oligo (X¹ _(PP)) is used to purify Part A. Thepart purification oligo (X¹ _(PP)) is then removed by melting, leavingthe part (Part A) with the linker oligonucleotide consisting of X¹ _(P2)and X¹ _(P1) attached.

Meanwhile, Part Z, which will bind to the 5′-end of Part A, is preparedin a similar manner to Part A. Part Z also has standard overhangs at the3′-end (S_(L)) and at the 5′-end (S_(P)) of the part.

In the part/linker assembly phase, the part (in this case Part Z) isligated and purified using one set of standard linker oligos. Theseoligos bind at the 3′-end of the part. The standard linker oligosinclude a partially double stranded linker oligonucleotide that consistsof a shorter oligonucleotide (X¹ _(L2)) that binds to the overhang S_(L)at the 3′-end of the part and a longer oligonucleotide (X¹ _(L1)) thatbinds directly to the 3′-end of the part on the strand which does nothave the overhang. X¹ _(L2) is complementary to X¹ _(L1) and since X¹_(L1) is longer than X¹ _(L2) a new overhang is created at the 3′-end ofthe part. This overhang is complementary to the overhang at the 5′-endof Part A that is formed from X¹ _(P)1

The standard linker oligos also include a linker purification oligo (X¹_(LP)) that binds to X¹ _(L2) and is partially complementary to X¹ _(L1)in the new region of overhang created when X¹ _(L2) binds to X¹ _(L1).The linker purification oligo (X¹ _(LP)) is used to purify Part Z. Thelinker purification oligo (X¹ _(LP)) is then removed by melting, leavingthe part (Part Z) with the linker oligonucleotide consisting of X¹ _(L2)and X¹ _(PL1) attached.

Part Z also has a set of standard part oligos, which bind at the 5′-endof the part. The standard part oligos include a partially doublestranded linker oligonucleotide that consists of a shorteroligonucleotide (X² _(P2)) that binds to the overhang S_(P) at the5′-end of the part and a longer oligonucleotide (X² _(P1)) that bindsdirectly to the 5′-end of the part on the strand which does not have theoverhang. X² _(P2) is complementary to X² _(P1) and since X² _(P1) islonger than X² _(P2) a new overhang is created at the 5′-end of thepart.

In the part assembly phase, the parts A and Z are ligated together. Theligation occurs by means of the complementarity of the overhangs at the3′-end of Part Z and at the 5′-end of Part A. These overhangs arecreated by oligos X¹ _(L1) and X¹ _(P1) respectively. It can be seenfrom FIG. 6 that the linker oligos of Part Z, X¹ _(L1) and X¹ _(L2), andthe part oligos of Part A, X¹ _(P2) and X¹ _(P1), together form astandard linker X¹. The standard linker X¹ is sometimes referred toherein as X1.

FIG. 6 demonstrates only the assembly of two parts, Part A and Part Z,but the method demonstrated in FIG. 6 can be used to produce an assemblywith a greater number of parts using the same process. In theseembodiments of the invention, standard linkers X², X³, X⁴, X⁵, X⁶ and soon will be formed (sometimes referred to herein as X², X³, X⁴, X⁵, X⁶and so on).

An alternative method for the assembly of a polynucleic acid sequenceaccording to the present invention is shown in FIG. 7.

In the part cloning phase, a truncated part (in this case truncated PartA) is prepared by carrying out PCR using a forward primer (A_(F)) and areverse primer (A_(R)). The primers are designed to produce a truncatedversion of Part A, lacking some of the sequence for the part, in thiscase the 5′-end sequence. As with the embodiment of the invention shownin FIG. 6, the truncated part is designed to be in a standard form, witha standard overhang at the 3′-end (S_(L)) and a standard overhang at the5′-end (A_(O)), each of which is designed to be recognised by aparticular restriction enzyme. Conveniently, the truncated part can becloned into a plasmid for storage before carrying out the partpreparation phase.

In the part preparation phase, the plasmid containing the truncated partis digested with one or more restriction enzymes that recognises andcuts the truncated part at a predefined sequence, leaving standardoverhangs at the 3′-end (S_(L)) and at the 5′-end (A_(O)) of thetruncated part.

In the part/linker assembly phase, the truncated part (in this casetruncated Part A) is ligated and purified using one set of standard partoligos. These oligos bind at the 5′-end of the truncated part. Thestandard part oligos include a partially double stranded linkeroligonucleotide that consists of a shorter oligonucleotide (A_(P2)) thatbinds to the overhang A_(O) at the 5′-end of the truncated part and alonger oligonucleotide (A_(P1)) that binds directly to the 5′-end of thetruncated part on the strand which does not have the overhang. A_(P2) iscomplementary to A_(P1) and since A_(P1) is longer than A_(P2) a newoverhang is created at the 5′-end of the part.

The standard part oligos also include a part purification oligo (A_(PP))that binds to A_(P2) and is partially complementary to A_(P1) in the newregion of overhang created when A_(P2) binds to A_(P1). The partpurification oligo (A_(PP)) is used to purify the truncated Part A. Thepart purification oligo (A_(PP)) is then removed by melting, leaving thetruncated part (Part A) with the linker oligonucleotide consisting ofA_(P2) and A_(P1) attached.

Meanwhile, truncated Part Z, which will bind to the 5′-end of truncatedPart A, is prepared in a similar manner to truncated Part A. TruncatedPart Z also has a standard overhang at the 3′-end (S_(L)). TruncatedPart Z also has an overhang at the 5′-end, which may be a standardoverhang (A_(O)) or some other overhang (e.g. Z_(O)).

In the part/linker assembly phase, the truncated part (in this case PartZ) is ligated and purified using one set of standard linker oligos.These oligos bind at the 3′-end of the truncated part. The standardlinker oligos include a partially double stranded linker oligonucleotidethat consists of a shorter oligonucleotide (A_(L2)) that binds to theoverhang S_(L) at the 3′-end of the truncated part and a longeroligonucleotide (A_(L1)) that binds directly to the 3′-end of thetruncated part on the strand which does not have the overhang. A_(L2) iscomplementary to A_(L1) and since A_(L1) is longer than A_(L2) a newoverhang is created at the 3′-end of the truncated part. This overhangis complementary to the overhang at the 5′-end of truncated Part A thatis formed from A_(P1).

The standard linker oligos also include a linker purification oligo(A_(LP)) that binds to A_(L2) and is partially complementary to A_(L1)in the new region of overhang created when A_(L2) binds to A_(L1). Thelinker purification oligo (A_(LP)) is used to purify Part Z. The linkerpurification oligo (A_(LP)) is then removed by melting, leaving thetruncated part (Part Z) with the linker oligonucleotide consisting ofA_(L2) and A_(pL1) attached.

Truncated Part Z also has a set of standard part oligos, which bind atthe 5′-end of the part. The standard part oligos include a partiallydouble stranded linker oligonucleotide that consists of a shorteroligonucleotide (Z_(P2)) that binds to the overhang at the 5′-end of thetruncated part, which may be a standard overhang (A_(O)) or some otheroverhang (e.g. Z_(O)), and a longer oligonucleotide (Z_(p1)) that bindsdirectly to the 5′-end of the truncated part on the strand which doesnot have the overhang. Z_(P2) is complementary to Z_(P1) and sinceZ_(P1) is longer than Z_(P2) a new overhang is created at the 5′-end ofthe truncated part.

In the part assembly phase, the truncated parts A and Z are ligatedtogether. The ligation occurs by means of the complementarity of theoverhangs at the 3′-end of truncated Part Z and at the 5′-end oftruncated Part A. These overhangs are created by oligos A_(L1) andA_(P1) respectively. It can be seen from FIG. 7 that in this embodimentof the invention the linker oligos of truncated Part Z, A_(L1) andA_(L2), and the part oligos of truncated Part A, A_(P2) and A_(P1),together with truncated Part A form the complete Part A sequence.

FIG. 7 demonstrates only the assembly of two truncated parts, truncatedPart A and truncated Part Z, but the method demonstrated in FIG. 7 canbe used to produce an assembly with a greater number of truncated partsusing the same process.

In the embodiment of the invention demonstrated in FIG. 6, the part isthe nucleic acid sequence with the standard overhangs at the 3′-end andat the 5′-end. This assembly process leads to a longer scar between theparts, the scar consisting of the standard linker X¹.

In the embodiment of the invention demonstrated in FIG. 7, the partincludes the truncated part and the accompanying linker and part oligos.This assembly process produces the non-truncated parts with a shortstandard scar (consisting of the overhangs at the 3′-end of each part,in this case S_(L)).

In one embodiment of the invention, the method involves preparing partswith short overhangs (typically of 3 or 4 bp) using one or morerestriction enzymes. One of the short overhangs must be the same in allthe parts; the other can be the same or can be different. Matchingoligos are then chosen that convert the short overhangs into longerunique overhangs. Pathway assembly is then carried out using the longunique overhangs.

If the sequence present in the oligos is viewed as a component of thepart (as in the embodiment demonstrated in FIG. 7), then the part isessentially truncated and then un-truncated during the assembly process.However, the part sequence can alternatively be viewed as not includingthe oligos (as in the embodiment demonstrated in FIG. 6). In this case,the assembly process involved adding an additional sequence (the linkerand part oligos) between the parts.

In one embodiment, the present invention provides a method for theassembly of a polynucleic acid sequence as shown in FIG. 10. Thisillustrates in more detail a method for the assembly of a polynucleicacid sequence that is shown schematically in FIG. 9.

FIG. 10 also includes a step prior to step (i) of the method of theinvention which involves ligating an oligonucleotide linker sequenceL1^(3′) to the 3′-end of a first nucleic acid sequence N1, ligating anoligonucleotide linker sequence L1^(5′) to the 5′-end of the firstnucleic acid sequence N1, ligating an oligonucleotide linker sequenceL2^(3′) to the 3′-end of the second nucleic acid sequence N2 andligating an oligonucleotide linker sequence L2^(5′) to the 5′-end of asecond nucleic acid sequence N2.

This step is referred to in FIG. 10 as “part ligation”. In this step,nucleic acid sequences are mixed with the relevant oligonucleotidelinker sequences and a ligase and incubated to allow ligation to occur.

In the embodiment illustrated in Example 1 herein, the first nucleicacid sequence N1 is the plasmid pSB1C3 and the second nucleic acidsequence N2 is DNA encoding either GFP or RFP. The pSB1C3 DNA, the GFPDNA and the RFP DNA is all pre-digested with the restriction enzymeEarI.

As shown in FIG. 9, for these reactions the oligonucleotide linkersequence L1^(3′) is called L2, the oligonucleotide linker sequenceL1^(5′) is called P1, the oligonucleotide linker sequence L2^(3′) iscalled L1 and the oligonucleotide linker sequence L2^(5′) is called P2.The oligonucleotide linker sequences P1, L1, P2 and L2 are collectivelyknown as part/linker oligos.

FIG. 10 includes two purification steps; these are referred to as “partpurification step 1” and “part purification step 2” in FIG. 10 and “partpurification 1” and “part purification 2” in FIG. 9. In the partpurification steps, biotinylated purification oligos PP1, PL2, PP2 andPL2 are used to remove any unbound oligonucleotide linker sequences. Thepurification oligos bind to the part/linker oligos described above. PP1binds to P1, PL2 binds to L2, PP2 binds to P2 and PL2 binds to L2. Inpart purification step 1, part purification oligos PP1 and PP2 are usedto remove any unbound oligonucleotide linker sequences P1 and P2. Inpart purification step 2, part purification oligos PL1 and PL2 are usedto remove any unbound oligonucleotide linker sequences L1 and L2.

Streptavidin coated magnetic beads are used in the purification steps.As mentioned above, the purification oligos are biotinylated, andtherefore bind to the streptavidin coated magnetic beads. Any unboundoligonucleotide linker sequences therefore bind to the purificationoligos, which in turn bind to the streptavidin coated magnetic beads,which can be separated from the reaction mixture by magnetic means.

A two-step purification method is also illustrated in FIG. 11.

The step referred to as “pathway assembly” in FIG. 10 corresponds tostep (iv) of the method of the invention. Thus, in this step the nucleicacid sequences are ligated to form a polynucleic acid sequence. As shownin FIG. 9, the assemblies that are formed are either pSB1C3.GFP orpSB1C3.RFP.

The final step shown in FIG. 10 is referred to as “transformation” (or“part transformation in FIG. 9). In this step, the polynucleic acidsequences formed in the pathway assembly step are transformed intosuitable cells, grown for a suitable amount of time and then plated outand results determined. A successful assembly of pSB1C3.GFP producesgreen cells and a successful assembly of pSB1C3.RFP produces red cells.The number of colonies (yield) and percent of colonies with correctphenotype (efficiency) can be determined for each of the assemblies.

FIG. 10 demonstrates only the assembly of two parts, but the methodshown in FIG. 10 can also be used to produce an assembly with a greaternumber of parts using the same process.

The method of the present invention has the following advantages:

-   -   All parts to be assembled are in a standard form.    -   A library of such standard parts can be created and any set of        those parts can be assembled in any order.    -   By using an offset cutter that leaves non-palidromic overhangs,        many incorrect side products arising from palindromic overhangs        (e.g. formed from most restriction enzymes) are eliminated.    -   No new oligos need to be synthesized during the assembly        process.    -   The process is extremely parallel and in the best case, requires        constant time independent of the number of parts being        assembled.    -   The entire time for assembly can be extremely fast. An optimal        assembly would only require two ligations and three biotin-based        purifications.    -   The entire process involves a small number of simple operations        amenable to automation.    -   The process does not require in vivo steps (e.g. yeast        recombination), enabling the construction of DNA that might be        unstable or toxic to cells.    -   The process is compatible with further in vivo processing (e.g.        bacterial transformation of assembled circular plasmid and        parts).    -   The only enzyme required in the assembly process is DNA ligase.        It does not require polymerases, nucleases, recombinases, or        other enzymes.    -   No amplification is required, reducing the chances of errors.    -   The resulting product is pure from incorrectly assembled        products, eliminating the costly need for sequencing, assuming        high quality input parts and oligos.    -   Ligation has been shown to scale to at least 150 kb.    -   Parts with similar overhangs can be assembled by splitting the        last pathway assembly step into multiple cycles (unlike, for        example, PCR based approaches).    -   Similar parts can be assembled in one pot if they are designed        appropriately. For example, there is flexibility when designing        how to break a part into the oligo portion and the rest. The        actual sequence of the overhang that will be ligated during the        one pot assembly step is entirely determined by the oligos used.        By changing the overhang (e.g. changing its length or whether        it's a 5′- or 3′-overhang), similar parts can be assembled in        one pot (e.g. assembling two GFP variants together).    -   A small number of parts can form a large number for pathways.        For example, suppose one has 5 possible promoters that one wants        to test in front of each of the genes in a 5 gene pathway. There        are 10 total parts (5 promoters+5 genes). The number of possible        promoter-gene and gene-promoter junctions is 50, i.e. at most 50        different part-linker fusions need to be done. These 50        part-linker fusions can then be assembled into 5⁵=3125 different        pathways in one pot reactions.

At least step (iv) of the method of the present invention is carried outon a microfluidic device.

As defined herein, a microfluidic device is a device for manipulatingminute amounts of fluid, usually microliters (μL or μl) or nanoliters(nL or nl). Such devices are known in the art. They are typicallysubstantially planar and frequently contain features such as chambers,channels and/or valves. Typically, when a microfluidic device contains aplurality of chambers, they are linked to each other via fluid channelswhich can contain valves. Such valves can be opened and closed in seriesto effect pumping of fluid through the fluid channels. Typically, thedimensions of such a microfluidic device are on the cm scale, forexample from around 5 cm to 8 cm by 6 cm to 11 cm, typically 5 cm by 11cm, 6 cm by 10 cm or 7 cm by 9 cm.

In one embodiment, the microfluidic device comprises at least one inputchamber, at least one storage chamber, at least one reaction chamber andat least one output chamber and wherein each of said at least one inputchamber, at least one storage chamber and at least one output chamber islinked by a separate fluid channel to said at least one reactionchamber.

An input chamber is a chamber for holding fluid which is open to theatmosphere such that fluid can be introduced into said chamber, forexample by injection. The input chamber can therefore also be describedas an injection chamber. The input chamber may optionally have aremovable cover, which is suitably transparent. An input chamber is alsodescribed herein as an “input reservoir”.

An output chamber is a chamber for holding fluid which is open to theatmosphere such that fluid can be removed from said chamber. The outputchamber can therefore also be described as a collection chamber. Theoutput chamber may optionally have a removable cover, which is suitablytransparent. An output chamber is also described herein as an “outputreservoir”.

A storage chamber is a chamber for holding fluid which is optionallyopen to the atmosphere such that fluid can be introduced into or removedfrom said chamber. The storage chamber may optionally have a removablecover, which is suitably transparent. A storage chamber is alsodescribed herein as a “temporary chamber” or an “intermediate productchamber”.

A reaction chamber is a chamber in which reactions, typically biologicalreactions, take place. The reaction chamber is typically closed to theatmosphere, typically in order to prevent contamination.

Each of the at least one input chamber, at least one storage chamber andat least one output chamber is linked by a separate fluid channel to theat least one reaction chamber. In other words, a fluid channel linkseach input chamber to each reaction chamber, a separate fluid channellinks each storage chamber to each reaction chamber and a separate fluidchannel links each output chamber to each reaction chamber. Thus, eachof the at least one input chamber, at least one storage chamber and atleast one output chamber is in fluid communication with the at least onereaction chamber.

A fluid channel is a vessel through which fluid can flow. Typically, thevolumes of fluid that flow through the fluid channel are on themicroliter or nanoliter scale.

In one embodiment, the microfluidic device comprises two input chambers,one storage chamber, one reaction chamber and two output chambers. Inthis embodiment, a fluid channel links the first input chamber to thereaction chamber, a second fluid channel links the second input chamberto the reaction chamber, a third fluid channel links the storage chamberto the reaction chamber, a fourth fluid channel links the first outputchamber to the reaction chamber and a fifth fluid channel links thesecond output chamber to the reaction chamber. An example of this layoutis illustrated in FIG. 12.

The input chambers, storage chamber and/or output chambers are typicallysubstantially circular. This embodiment is illustrated in FIG. 12.

In one embodiment, the reaction chamber is substantially elliptical.This embodiment is illustrated in FIG. 12.

In one embodiment, one or more of the fluid channels has one or morevalves. These valves are operable to control the flow of fluid throughthe fluid channels. As such, the valves can prevent or allow the flow offluid through the fluid channels. Typically, one or more of the fluidchannels has two or more valves, such as three, four or five valves. Asset out above, such valves can be opened and closed in series to effectpumping of fluid through the fluid channels.

In one embodiment, one or more of the fluid channels has two valves anda pump chamber is located between these two valves. A pump chamber is achamber for holding fluid and is typically closed to the atmosphere. Thepump chamber is typically substantially circular. In one embodiment,more than one of the fluid channels has this arrangement of valves and apump chamber. For example, two, three, four or more of the fluidchannels has this arrangement of valves and a pump chamber. As shown inFIG. 13, the arrangement of two valves and a pump chamber is describedherein as a pump.

As shown in FIG. 13, the arrangement of one input chamber, together withits respective fluid channel containing two valves with a pump chamberinbetween is described herein as an “inlet channel” or “input channel”.Similarly, as shown in FIG. 13, the arrangement of the storage chamber,together with its respective fluid channel containing two valves with apump chamber inbetween is described herein as an “intermediate productchannel” or “storage channel”. As shown in FIG. 13, the arrangement ofan output chamber together with its respective fluid channel containinga valve is described herein as a “waste and/or product chamber”.

Typically, the depth of the chambers and fluid channels in themicrofluidic device is in the range from 100 μm to 500 μm, typicallyfrom 200 μm to 400 μm, more typically from 250 μm to 300 μm. Typically,the depth of the chambers and fluid channels in the microfluidic deviceis 250 μm.

Typically, the diameter of the input chamber, storage chamber and/oroutput chamber is in the range from 1 mm to 10 mm, typically from 2 mmto 9 mm, from 3 mm to 8 mm, from 4 mm to 7 mm, from 4.5 mm to 6 mm,typically from 4.5 mm to 5.5 mm, from 4.6 mm to 5.4 mm, from 4.7 mm to5.3 mm or from 4.8 mm to 5.2 mm, typically 5.2 mm.

Typically, the dimensions of the reaction chamber are from 3 mm to 9 mmby 5 mm to 15 mm, for example 3 mm by 15 mm, 4 mm by 14 mm, 5 mm by 13mm, 6 mm by 12 mm, 7 mm by 11 mm. Typically, the dimensions of thereaction chamber are around 6 mm by 10.5 mm, typically 5.9 mm by 10.4mm.

Typically, the width of the fluid channel is in the range from 0.1 mm to0.8 mm, typically from 0.2 mm to 0.7 mm, from 0.3 mm to 0.6 mm, from 0.4mm to 0.5 mm, typically 0.4 mm.

Typically, the diameter of the pump chamber is in the range from 1 mm to5 mm, typically from 2 mm to 4 mm, from 2.5 mm to 3.5 mm, from 2.75 mmto 3.25 mm, typically around 3.16 mm.

Typically, the dimensions of the valves are from 0.5 mm to 1.5 mm by 2mm to 4 mm, for example 0.5 mm by 4 mm, 1 mm by 3 mm, 1.5 mm by 2 mm,typically 1 mm by 2.2 mm.

In one embodiment of the invention, the microfluidic device comprisestwo input chambers, one storage chamber, one reaction chamber and twooutput chambers, and each of the fluid channels linking the inputchambers to the reaction chamber and the fluid channel linking thestorage chamber to the reaction chamber have two valves and a pumpchamber is located between these two valves. In addition, each of thefluid channels linking the output chambers to the reaction chamber hasone valve. This embodiment of the invention is illustrated in FIG. 12.

Such a microfluidic device is suitable for carrying out the methoddescribed in Example 1 herein. Nucleic acid sequences are typicallyintroduced into the input chambers together with the necessary reagents.The nucleic acid sequences and reagents are pumped into the centralreaction chamber where ligation takes place and the products, i.e.polynucleic acid sequences having the desired assemblies, are pumped tothe output chamber and then removed.

As set out above, at least step (iv) of the method of the presentinvention is carried out on a microfluidic device. However, other stepsof the method of the present invention can also be carried out on amicrofluidic device. For example, the step of purifying the nucleic acidsequences immediately prior to step (iv) can also be carried out on amicrofluidic device. In this embodiment of the invention, step (iv) andthe step of purifying the nucleic acid sequences immediately prior tostep (iv) can be carried out on the same microfluidic device ordifferent microfluidic devices. When these steps are carried out ondifferent microfluidic devices, the microfluidic devices are optionallyconnected, or if not connected, means are provided for transferringfluid from one device to another.

The microfluidic device shown in FIG. 12 is suitable for carrying outboth step (iv) and the step of purifying the nucleic acid sequencesimmediately prior to step (iv). Thus, in this embodiment of theinvention, step (iv) and the step of purifying the nucleic acidsequences immediately prior to step (iv) are carried out on the samemicrofluidic device.

In the embodiment of the invention where step (iv) and the step ofpurifying the nucleic acid sequences immediately prior to step (iv) arecarried out on different microfluidic devices, the layout of themicrofluidic devices used for each step can be tailored to therequirements of each of such steps.

Therefore, in one embodiment, the microfluidic device is suitable forcarrying out step (iv). This corresponds to the step labelled “pathwayassembly” in FIG. 10. Such a microfluidic device is also referred to asan “assembly chip” herein. Such a microfluidic device allows the highthroughput assembly of polynucleic acid sequences such as DNA sequencesto be carried out using the method of the invention. A microfluidicdevice suitable for carrying out step (iv) of the method of theinvention can also be suitable for carrying out the step referred to inFIG. 10 as “part ligation”. This is a step prior to step (i) of themethod of the invention which involves ligating an oligonucleotidelinker sequence L1^(3′) to the 3′-end of a first nucleic acid sequenceN1 and/or ligating an oligonucleotide linker sequence L1^(5′) to the5′-end of the first nucleic acid sequence N1 and/or ligating anoligonucleotide linker sequence L2^(3′) to the 3′-end of the secondnucleic acid sequence N2 and/or ligating an oligonucleotide linkersequence L2^(5′) to the 5′-end of a second nucleic acid sequence N2.

In this embodiment of the invention, the microfluidic device comprisesat least two input chambers, at least one auxiliary chamber and at leastone output chamber and each of said at least two input chambers and saidat least one auxiliary chamber is linked by a central fluid channel tosaid at least one output chamber. A microfluidic device having a layoutin accordance with this embodiment of the invention is shown in FIG.14A.

An auxiliary chamber is a chamber for holding fluid which is open to theatmosphere such that fluid can be introduced into said chamber, forexample by injection. The auxiliary chamber may optionally have aremovable cover, which is suitably transparent. An auxiliary chamber isalso described herein as an “auxiliary reservoir”.

In this embodiment of the invention, each of the nucleic acid sequencesto be assembled into a polynucleic acid sequence by the method of theinvention is typically introduced into a separate input chamber. Thereis therefore typically one input chamber per nucleic acid sequence thatis assembled into a polynucleic acid sequence by the method of theinvention. The number of input chambers is typically 2, 3, 4, 5, 6, 7,8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 but can be more than20, for example 25, 30, 35, 40, 45, 48, 50, 52, 54, 56, 58 or 60.

In one embodiment, half of the input chambers lie on one side of thecentral fluid channel and half of the input chambers lie on the otherside of the central fluid channel. Alternatively, there may be anasymmetric arrangement of input chambers, i.e. more input chambers lieon one side of the central fluid channel than the other.

In this embodiment of the invention, there are typically 1, 2, 3, 4, 5or 6 auxiliary chambers. Typically, there are 2 auxiliary chambers.

In this embodiment of the invention, there are typically 1, 2, 3, 4, 5or 6 output chambers. Typically, there are 2 output chambers.

All of the input chambers feed into the central fluid channel whichlinks each of the input chambers to the output chamber. In operation, afluid such as oil or water can be introduced into the auxiliary chamberand then pumped into the central fluid channel, which typically has theeffect of pushing the nucleic acid sequences along the central fluidchannel where they mix and ligate, and eventually arrive in the outputchambers, from which the finished polynucleic acid product can beremoved. Suitably, a fluid such as oil can be used to push the nucleicacid sequences along the channels in order to avoid dilution. Water mayalso be used for washing or other purposes.

The central fluid channel is typically straight, as shown in FIG. 14A.However, the central fluid channel may take another suitableconfiguration and thus may be, for example, curved or in the form of azig zag.

In one embodiment of the invention, one or more of the input chambers islinked to the central fluid channel by a further fluid channel.Typically, each of the input chambers is linked to the central fluidchannel by a further fluid channel. In one embodiment, one or more ofthe further fluid channels has one or more valves. In one embodiment,one or more of such further fluid channels has two valves and a pumpchamber is located between these two valves. In one embodiment, each ofthe further fluid channels has two valves and a pump chamber is locatedbetween these two valves. An example of this embodiment of the inventionis illustrated in FIG. 14A.

In one embodiment, the further fluid channels linking input chambers oneither side of the central fluid channel to the central fluid channelare lined up symmetrically, as shown in FIG. 14A. In other words, thefluid channels linking input chambers on either side of the centralfluid channel to the central fluid channel enter the central fluidchannel at a position directly opposite to each other. In anotherembodiment, the further fluid channels linking input chambers on eitherside of the central fluid channel to the central fluid channel are linedup asymmetrically, as shown in FIG. 14B. In other words, the connectionsof the further fluid channels to either side of the central fluidchannel are offset. This layout can be used, for example, to reducecross-contamination.

In one embodiment, the tips of the further fluid channels linking theinput chambers to the central fluid channel are nozzled, as shown inFIG. 14C. In this embodiment, the microfluidic device is typicallymanufactured by laser cutting.

In one embodiment of the invention, one or more of the auxiliarychambers is linked to the central fluid channel by a further fluidchannel. Typically, each of the auxiliary chambers is linked to thecentral fluid channel by a further fluid channel. In one embodiment, oneor more of the further fluid channels has one or more valves. In oneembodiment, one or more of such further fluid channels has two valvesand a pump chamber is located between these two valves. In oneembodiment, each of the further fluid channels has two valves and a pumpchamber is located between these two valves. An example of thisembodiment of the invention is illustrated in FIG. 14A.

In one embodiment of the invention, one or more of the output chambersis linked to the central fluid channel by a further fluid channel.Typically, each of the output chambers is linked to the central fluidchannel by a further fluid channel. In one embodiment, one or more ofthe further fluid channels has one or more valves. An example of thisembodiment of the invention is illustrated in FIG. 14A.

In one embodiment, the microfluidic device further comprises a reactionchamber.

In one embodiment, the microfluidic device comprises 16 input chambers,2 auxiliary chambers and 2 output chambers. An example of this layout isillustrated in FIG. 14A. This layout is also referred to herein as “Chip3A”. Using this microfluidic device, nucleic acid sequences to beassembled can be placed in the input chambers in order to be pumped in acombinatorial fashion, and pushed via fluid pumping to the outputchamber, ready to be collected in the form of assembled polynucleic acidsequences. As set out above, this microfluidic device can also be usedto carry out a ligation step prior to step (i) of the method of theinvention such as that referred to in FIG. 10 as “part ligation”. Thismicrofluidic device can therefore be used to carry out high throughputligation or assembly of up to 16 inputs, typically nucleic acidsequences, in a single device.

In another embodiment, the microfluidic device is suitable for carryingout the step of purifying the nucleic acid sequences immediately priorto step (iv). In one embodiment, multiple purification steps are carriedout immediately prior to step (iv). Typically, two purification stepsare carried out immediately prior to step (iv).

In this embodiment of the invention, the microfluidic device comprisesat least one input chamber, at least one auxiliary chamber, at least onereaction chamber, at least one waste chamber and at least one outputchamber, wherein said at least one input chamber is linked by a fluidchannel to said at least one reaction chamber, said at least onereaction chamber is linked by a fluid channel to said at least oneoutput chamber, said at least one auxiliary chamber is linked by a fluidchannel to said at least one waste chamber, and wherein the fluidchannel linking said at least one auxiliary chamber to said at least onewaste chamber intersects the fluid channel linking said at least oneinput chamber to said at least one reaction chamber.

A waste chamber is a chamber for holding fluid which is open to theatmosphere such that fluid can be removed from said chamber. The wastechamber may optionally have a removable cover, which is suitablytransparent. A waste chamber is also described herein as a “wastereservoir”.

In this embodiment of the invention, there is typically one inputchamber per nucleic acid sequence that is assembled into a polynucleicacid sequence by the method of the invention.

The number of input chambers is typically 2, 3, 4, 5, 6, 7, 8, 9, 10,11, 12, 13, 14, 15, 16, 17, 18, 19 or 20 but can be more than 20, forexample 25, 30, 35, 40, 45, 48, 50, 52, 54, 56, 58 or 60.

The number of input chambers is typically the same as the number ofreaction chambers. The number of input chambers is also typically thesame as the number of output chambers. The number of reaction chambersis also typically the same as the number of output chambers. The numberof output chambers and the number of reaction chambers is therefore alsotypically 2, 3, 4, 5, 6, 7, 8, 9, 10, 11, 12, 13, 14, 15, 16, 17, 18, 19or 20 but can be more than 20, for example 25, 30, 35, 40, 45, 48, 50,52, 54, 56, 58 or 60.

In this embodiment, the fluid channel linking the at least one auxiliarychamber to the at least one waste chamber intersects the fluid channellinking said at least one input chamber to said at least one reactionchamber. In other words, the fluid channel linking the at least oneauxiliary chamber to the at least one waste chamber crosses over orjoins the fluid channel linking said at least one input chamber to saidat least one reaction chamber. Typically, there is one auxiliary chamberand one waste chamber. In one embodiment, where there are a plurality ofinput chambers, the fluid channel linking the auxiliary chamber to thewaste chamber intersects each of the fluid channels linking the inputchambers to the reaction chambers. Typically, the fluid channel linkingthe auxiliary chamber to the waste chamber intersects each of the fluidchannels linking the input chambers to the reaction chambers at an angleof 90°. This embodiment is illustrated in FIG. 15.

In one embodiment, one or more of the fluid channels linking the inputchambers to the reaction chambers has two valves and a pump chamber islocated between these two valves. In one embodiment, the fluid channellinking the at least one auxiliary chamber to the at least one wastechamber intersects one or more of the fluid channels linking the atleast one input chamber to the at least one reaction chamber between oneof the valves and the pump chamber. In other embodiments, the fluidchannel linking the at least one auxiliary chamber to the at least onewaste chamber intersects one or more of the fluid channels linking theat least one input chamber to the at least one reaction chamber betweenthe input chamber and one of the valves, or between one of the valvesand the reaction chamber.

In one embodiment, there is an additional fluid channel that branchesfrom the fluid channel that links the input chamber to the reactionchamber and joins the fluid channel that links the reaction chamber tothe output chamber. This fluid channel allows fluid flowing from theinput chamber to the output chamber to bypass the reaction chamber. Thisfluid channel can therefore be described as a bypass fluid channel. Thisfluid channel typically includes two valves and a pump chamber locatedbetween these two valves. This fluid channel can therefore act as a sidepump, which can be used, for example, for mixing fluid. In oneembodiment, such an additional fluid channel bypasses each of thereaction chambers.

In one embodiment, the microfluidic device comprises 4 input chambers, 1auxiliary chamber, 4 reaction chambers, 1 waste chamber and 4 outputchambers and the fluid channel linking the auxiliary chamber to thewaste chamber intersects each of the fluid channels linking the inputchambers to the reaction chambers. An example of this embodiment of theinvention, is illustrated in FIG. 15. Heating and/or magnetic actuation(separation and/or mixing) can be carried out on this microfluidicdevice as described herein. For example, heating can be carried outusing a Peltier device.

In one embodiment, the layout of the microfluidic device described inrelation to this embodiment of the invention can be reproduced twice ormore on the same microfluidic device. An example of a microfluidicdevice wherein the above-described layout is reproduced twice isillustrated in FIG. 15. This layout is also referred to herein as “Chip3P”. The same layout can therefore be reproduced for example 2, 3, 4, 5,6 or more times on the same microfluidic device. This allows forpurification of various nucleic acid sequences in parallel and is usefulin a high throughput method. Alternatively or in addition, differentlayouts can be included in the same microfluidic device such thatligation and purification can be carried out on the same microfluidicdevice, but in different areas of the device.

FIG. 27 demonstrates in detail an example of how to operate a pump toflow liquid from a chamber through a fluid channel using themicrofluidic device of the invention. For example, the operation of thepump demonstrated in FIG. 27 can be used to pump liquid from one of theinput chambers into the reaction chamber or from the storage chamber tothe reaction chamber in the microfluidic device shown in FIG. 12.Alternatively, the operation of the pump demonstrated in FIG. 27 can beused to pump liquid from one of the input chambers into the centralfluid channel or from one of the auxiliary chambers into the centralfluid channel in the microfluidic device shown in FIG. 14A.Alternatively, the operation of the pump demonstrated in FIG. 27 can beused to pump liquid from one of the input chambers into the one of thereaction chambers, from one of the reaction chambers to one of theoutput chambers, from one of the auxiliary chambers to one of the wastechambers or through the side pump in the microfluidic device shown inFIG. 15.

Each “pump” unit features two valves and 1 pump chamber. The pumpchamber is surrounded by two valves in all pump unit arrangements.Different pump sequences can be used to activate the flow in thechannel. FIG. 27 shows the order in which the valves and the pumpchamber should be closed in order to effect flow of fluid in the fluidchannel in the direction shown in the Figure. In FIG. 27 the sequenceused corresponds to: 100, 110, 010, 011, 001, 101, where “1” correspondsto a structure closed (valve or pump chamber) and “0” corresponds to anopen structure. Note that this sequence is not the only one that couldbe used.

By this operation a flow is actuated. This is not dissimilar to theactuation of a peristaltic pump. However, the dimensions are muchsmaller and a decoupling concept is added. These six sequencescorrespond to a single pumping cycle. Pumping can also be effected inthe opposite direction to that shown in FIG. 27.

For the microfluidic device shown in FIG. 12, in some embodiments, thepumps between both of the input chambers and the reaction chamber areactivated at the same time and in other embodiments only one of thepumps between the input chambers and the reaction chamber is used at atime. In other words, in the latter embodiment, while one pump isactivated, the other pump remains closed at all times. This latterembodiment was carried out in Example 1 herein.

FIG. 28 presents some of the different fluidic cycles that can beperformed on the microfluidic device. These cycles do not necessarilyrepresent a chronological order. However, they represent some of the“movements” which can be performed on-chip. FIG. 10, which describes theprotocol used in Example 1 herein, also refers in the last column to thechip cycles referred to in FIG. 28, using the same lettering. FIG. 28demonstrates the fluidic cycles on the microfluidic device shown inFIGS. 12 and 13, but is equally applicable to the other microfluidicdevices of the invention described herein.

The numbers in FIG. 28 represent valves. As can be seen from FIG. 28,the microfluidic device shown in that Figure has 12 valves. In thisembodiment, the reaction chamber has a valve. This is valve number 10 inFIG. 28.

Each pump “actuation” corresponds to a number n of pumping cycles aspresented elsewhere.

The fluidic steps shown in FIG. 28 are as follows:

-   -   a—Fluid from inlet channel 1 is pumped into the Reaction        chamber. Valve 11 is left open to allow the fluid out into        Waste/product chamber 1. Inlet Channel 2 and Intermediate        product channel are closed. Biological example: Load magnetic        beads and binding buffer into the reaction chamber in part        purification step 1 shown in FIG. 10.    -   b—Inlet channel 1 and Intermediate product channel are closed.        Fluid from the inlet channel 2 is loaded into the reaction        chamber. Biological example: Oligos and binding buffer are        loaded into the reaction chamber to bind on the magnetic beads        in part purification step 1 shown in FIG. 10.    -   c—All the valves are closed. The reaction chamber is therefore        isolated. A manual mixing protocol (see elsewhere) is applied on        the chamber. Biological example: Oligos and beads are mixed        together for better binding in part purification step 1 and part        purification step 2 shown in FIG. 10.    -   d—Inlet channel 2 is closed, valves 11 and 12 are closed.        Intermediate product channel and inlet channel 1 are open. Inlet        channel 1 is pumped forward (ie from input chamber to reaction        chamber) and Intermediate product channel is pumped backward (ie        from reaction chamber to storage chamber) in order to push the        fluid inside the reaction chamber with a fresh fluid from Inlet        channel 1. Biological example: Elution from part purification        step 1 is stored in the intermediate product channel.    -   e—All channels closed but Inlet channel 2. A reagent is        introduced in the reaction chamber from inlet channel 2. Valve        11 is opened for evacuation of the previous fluid from the        reaction chamber. Biological example: Oligos and binding buffer        are loaded into the reaction chamber in part purification step 2        shown in FIG. 10.    -   f—All channels closed but Intermediate product channel. The        product stored in the Intermediate product channel is pumped        back into the reaction chamber. Biological example: Elution from        part purification step 1 is introduced in part purification step        2.    -   g—All the valves are closed. The reaction chamber is therefore        isolated. A heating protocol (see elsewhere) is applied on the        chamber. Biological example: Heating to elute oligos from        magnetic beads (unbinding) in part purification step 1 and part        purification step 2 shown in FIG. 10.    -   h—The reaction chamber is emptied into the waste and/or product        chamber number 1 via the actuation of a buffer flow in inlet        channel number 1. Biological example: Unloading of the elution        product.

Two exemplary techniques to manufacture microfluidic structures (width<1mm) in PMMA substrates with stretched silicone rubber membrane to closethe structure are as follows. The first technique involves one layer ofdouble-sided adhesive transfer tape, and the second technique involvestwo layers of double-sided adhesive transfer tape.

In the first technique (FIG. 16) the microchannels are engraved directlyon to the PMMA block coated with a layer of double-sided transfer tapeprotected on one side. After engraving of the microchannels theprotective layer is removed and the stretched silicone rubber membranecan be directly applied to seal the channel.

In the second technique (FIG. 17), the channels are cut through a thinlayer of PMMA sheet coated with 2 layers of double-sided adhesivetransfer tape using laser cutting. The microfluidic access are laser-cutin the PMMA substrate. One protective sheet is removed from the cutthrough piece and applied to the PMMA substrate. The bond can beenhanced by simply manually exerting pressure on the assembly. This canalso be done by using a roller. The second protective cover is thenremoved and the stretched silicone membrane is bonded onto the assembly.

In one embodiment, the chambers and the fluid channels are locatedbetween a rigid layer and an elastic layer and the microfluidic deviceis configured so that deformation of the elastic layer manipulates fluidif present in said chambers or said fluid channels.

This embodiment of the present invention is described in detail inInternational Patent Application No. PCT/GB2009/002968 (WO 2010/073020).

The microfluidic device can be detachably coupleable to a controlplatform that is operable to deform the elastic layer.

The elastic layer can form at least part of an external surface of themicrofluidic device. At least one part of the elastic layer can bedeformable to cause operation of a microfluidic control component of themicrofluidic device. The microfluidic control component can comprise avalve, mixer, or pump.

In one embodiment, the microfluidic device is part of a microfluidicsystem comprising a microfluidic device as defined herein; and a controlplatform comprising means for deforming the elastic layer thereby tomanipulate fluid in the at least one fluid chamber or channel.

By providing a control platform and a detachable microfluidic device, asystem that is particularly simple to manufacture can be provided. Theelastic layer can provide an interface that enables the manipulation offluid in the at least one fluid chamber or channel using devicesexternal of the microfluidic device, provided for example on the controlplatform.

This embodiment of the present invention is also described in detail inInternational Patent Application No. PCT/GB2009/002968 (WO 2010/073020).

Electronic, electromechanical, optical or other complex control ormeasurement components can be provided on the control platform ratherthan on the microfluidic device, reducing the complexity of manufactureof the microfluidic device. The microfluidic device can be manufacturedusing low cost materials and fabrication processes, and can be treatedas disposable.

The manipulation of fluid in the at least one fluid chamber of channelcan include, for example, controlling fluid flow or other fluiddynamics. Fluid flow can be flow along a chamber or channel or fluidflow within a chamber or channel.

A microfluidic system or device can be for example a system or devicefor manipulation of fluids on the millimeter or sub-millimeter scale,for example a system or device that includes at least one fluid chamberor channel at least part of which has at least one dimension that isless than or equal to around 1 mm.

The rigid layer is usually sufficiently rigid that if the rigid layer isheld stationary, a force applied to the elastic layer causes adeformation of the elastic layer relative to the rigid layer. The rigidlayer can be substantially rigid in whole or part. The rigid layer cancomprise a plurality of sub-layers or components. The rigid layer canfor example comprise a substantially rigid portion (for example asubstantially rigid frame) and a flexible portion fixed to thesubstantially rigid portion.

The microfluidic device and the detachable control platform can becoupleable in at least one alignment position, in which the means fordeforming the elastic layer is operable to selectively deform at leastone selected part of the elastic layer. Thus, fluid can be manipulatedat selected parts of the microfluidic device.

The at least one selected part of the elastic layer can comprise atleast one part of the elastic layer at which deformation of the elasticlayer causes operation of a microfluidic control component of themicrofluidic device. The microfluidic control component can comprise avalve, mixer, or pump.

The microfluidic device and the control platform can be coupleable in aplurality of different alignment positions, and in each alignmentposition deformation of the elastic layer can cause operation of arespective at least one microfluidic control component of themicrofluidic device. Thus, the same control platform can be used toperform a plurality of different operations on fluid in the microfluidicdevice. The microfluidic device can be placed in the plurality ofdifferent alignment positions in turn, with a different operation beingperformed on fluid in the microfluidic device at each alignmentposition.

The means for deforming the elastic layer can comprise means forapplying force. The control platform can comprise an external face thatis coupleable to the elastic layer of the microfluidic device, and themeans for applying force can be operable to apply force at at least onepart of the external face. The external face can be in contact with, orspaced apart from, the elastic layer of the microfluidic device. Themeans for applying force can be operable to apply a force that has acomponent in a direction substantially perpendicular to the externalface.

The means for applying force can be operable to apply force over anarea, that area being larger than the area of the at least one part ofthe elastic layer at which deformation of the elastic layer causesoperation of a microfluidic control component of the microfluidicdevice. Thus, it can be particularly straightforward to align thecontrol platform and the microfluidic device, as it can be sufficientthat the larger area over which force is applied covers the smaller areaof the at least one part of the elastic layer at which deformation ofthe elastic layer causes operation of a microfluidic control componentof the microfluidic device. That feature is particularly useful when themeans for applying force is able to cause different amounts ofdeformation of the elastic layer across the area over which force isapplied, for example when the means for applying force applies forceusing fluid pressure.

The means for deforming the elastic layer can comprise means forapplying fluid pressure to the elastic layer. The means for applyingfluid pressure can be operable to apply pressure to one side of theelastic layer that is greater than or less than the pressure acting onthe other side of the elastic layer. The applied pressure can be anover-pressure or an under-pressure, and can comprise an at least partialvacuum. The means for applying fluid pressure can be arranged to providepressurised fluid in direct contact with the elastic layer.

The means for deforming the elastic layer can comprise a microactuatormechanism.

The elastic layer can form at least part of an external surface of themicrofluidic device.

The system can further comprise alignment means for aligning the controlplatform and the microfluidic device. The alignment means can beconfigured to align the or a face of the control platform with theelastic layer. The alignment means can be operable to align the controlplatform and the microfluidic device in the or an at least one alignmentposition. The alignment means can be operable to align the means fordeforming the elastic layer with the or an at least one selected part ofthe elastic layer.

The alignment means can be arranged to align the control platform andthe microfluidic device so that the area over which the means forapplying force is operable to apply force at least partially overlapsthe at least one part of the elastic layer at which deformation of theelastic layer causes operation of a microfluidic control component ofthe microfluidic device. The alignment means can be operable to alignthe face and elastic layer to be substantially parallel.

The alignment means can comprise at least one male element and at leastone female element configured to receive the at least one male element.The male element or at least one of the male elements can be provided onone of the microfluidic device and the control platform and thecorresponding female element or a corresponding at least one of thefemale elements can be provided on the other of the microfluidic deviceand the control platform. The alignment means can comprise at least onealignment mark on each of the microfluidic device and the controlplatform.

The system can further comprise means for detachably coupling thecontrol platform to the microfluidic device. The coupling means cancomprise means for forming a seal between at least part of the controlplatform and at least part of the elastic layer.

The means for deforming the elastic layer can comprise means forapplying fluid pressure to the elastic layer, and the means for forminga seal can be arranged to form a seal around an area of the elasticlayer so that fluid pressure is applied to the elastic layer over thesealed area. The sealed area can comprise the or an at least one part ofthe elastic layer at which deformation of the elastic layer causesoperation of a microfluidic control component of the microfluidicdevice. The sealed area can be greater than the area of the or an atleast one part of the elastic layer at which deformation of the elasticlayer causes operation of a microfluidic control component of themicrofluidic device.

The control platform can comprise the or a face that is coupleable tothe elastic layer of the microfluidic layer, and the means for forming aseal can comprise at least one element that protrudes above the face ofthe control platform for engagement with the elastic layer.

The means for forming a seal can comprise an O-ring. The coupling meanscan comprise fixing means for detachably fixing the microfluidic deviceto the control platform, for example at least one of a clamp, a screw, abolt and an adhesive, typically a releasable adhesive.

The control platform can comprise at least one device for performing anoperation on fluid in the microfluidic device. The at least one devicefor performing an operation can comprise at least one sensor for sensinga property of fluid in the microfluidic device, or can comprise a devicefor altering a property of the fluid, for example a heater. Typically,the heater is a Peltier device.

The system can further comprise biasing means for biasing away from thecontrol platform the at least one device for performing an operation.The biasing means can be arranged so that when the microfluidic deviceand the control platform are coupled such that the deforming means isoperable to deform the elastic layer, the device for performing anoperation on the fluid is biased towards the microfluidic device. Thebiasing means can be arranged so that when the microfluidic device andthe control platform are coupled such that the deforming means isoperable to deform the elastic layer, the device for performing anoperation on the fluid is biased to be in contact with the elasticlayer. The biasing means can comprise at least one spring.

The system can further comprise a plurality of microfluidic devices eachof which is coupleable to the control platform. The system can comprisea plurality of control platforms, each of which is coupleable to themicrofluidic device or to each of the microfluidic devices.

The system can further comprise means for controlling operation of thedeforming means.

The elastic layer can form a wall of the fluid chamber or channel, andthe fluid chamber or channel can comprise a further, opposing wall, andthe control means can be operable to control the deforming means todeform the elastic layer towards the opposing wall. The control meanscan be operable to control the deforming means to deform the elasticlayer to be in contact with the opposing wall.

The control means can be operable to successively deform different partsof the elastic layer in a sequence to perform a desired fluid operation.The desired fluid operation can comprise at least one of pumping, mixingand allowing or preventing flow of the fluid. The control means can beoperable to repeatedly deform the or an at least part of the elasticlayer thereby to perform a fluid operation.

In one embodiment, the microfluidic system comprises a microfluidicdevice comprising at least one fluid chamber or channel, wherein anelastic layer forms at least one wall of the at least one fluid chamberor channel; means for deforming the elastic layer; control meansoperable to control the deforming means to repeatedly deform the elasticlayer thereby to perform an operation on fluid in the fluid chamber orchannel. The operation can comprise a mixing operation or a pumpingoperation. The control means can be operable to control the rate ofrepetition of deformation of the elastic layer. The control means canthereby control the amplitude of deformation of the elastic layer and/orthe rate of pumping or mixing. The control means can be operable tocontrol the rate of repetition of deformation of the elastic layer to begreater than a resonant frequency of vibration of the elastic layer.

In one embodiment, there is provided a method of manipulating fluid in amicrofluidic system comprising coupling to a control platform adetachable microfluidic device comprising a rigid layer, an elasticlayer and at least one fluid chamber or channel between the rigid layerand the elastic layer, and deforming the elastic layer thereby tomanipulate fluid in the at least one fluid chamber or channel.

In one embodiment, there is provided a method of performing an operationon a fluid in at least one fluid chamber or channel of a microfluidicdevice, wherein an elastic layer forms at least one wall of the at leastone fluid chamber or channel, the method comprising repeatedly deformingthe elastic layer. The method can comprise repeatedly deforming theelastic layer and controlling the rate of repetition of deformation ofthe elastic layer to be greater than a resonant frequency of vibrationof the elastic layer.

The invention can provide for the manipulation of fluids withinmicrofluidic devices by means of micromechanical movements of atwo-layer hybrid material, driven by a decoupled microscopicservomechanism. By a decoupled microscopic servomechanism is meant amicroscopic servomechanism that is not integrated into the two-layerhybrid material. The decoupled microscopic servomechanism may becoupleable to the two-layer material.

An integrated microfluidic system including a disposable microfluidicdevice and a microfluidic control plate can be provided. A decoupledcontrol unit can be provided. A system and manufacturing method fordecoupled micromechanical manipulation and control of fluid flow andfluid dynamics in a microfluidic device can be provided.

The following features can be provided: 1.) two-layer hybridmicrofluidic device combining a rigid polymer or other rigid layer and adeformable elastic membrane, therein to be used as a microvalve, and/ora micropump, and/or a micromixer, and 2.) decoupling of the microfluidicdevice from the microactuator mechanism. The microactuator mechanism canbe detached from the microfluidic chip, and the microactuator mechanismcan instead be housed in a separate but aligned underlying controlplatform. Such a design can combine the use of low-cost, disposablemicrofluidic devices with a re-useable microactuator-based fluid controlplatform, giving the potential for use in a wide range of lab-on-a-chipapplications.

Microfluidic flow control may be achieved through combined use of atwo-layer hybrid microfluidic device and an array of microactuators on acontrol platform.

FIGS. 33a and 33b show a microfluidic device 2 according to oneembodiment. The microfluidic device 2 has a two layer structurecomprising a first, rigid layer 4, and second deformable, elastic layer6. In the embodiment of FIGS. 33a and 33b , the rigid layer 4 is formedof lithographically patterned SU-8 polymer 5 spin coated on a glasssubstrate 7. The dimensions of the rigid layer are 1 mm (width) by 5 mm(length) by 100 μm (thickness), and the elastic layer is formed of asilicone film of thickness 100 μm. The rigid layer 4 is fabricated tocontain fluid flow chambers and/or channels. A single channel 8 of width200 μm and depth 100 μm is shown in cross-section in FIGS. 33a and 33 b.

The rigid and elastic layers have equivalent length and width, and areaffixed together creating the two-layer device 2. First a layer of SU-8resin is spin coated on the glass substrate 7. The thickness of the SU-8can be precisely controlled by the spin rate. After the SU-8 is exposedto UV radiation through a mask, well-defined microstructures can beformed where the exposed area is cross-linked and unexposed area iswashed away by solvent. A uniform layer of epoxy adhesive is thenapplied on the surface of the SU-8 resin, to which the elastic layer 6is then adhered. Alternatively, oxygen plasma can be used to create achemically reactive surface of the microfluidic device before elasticfilm is press bonded to the microfluidic device.

As shown in FIG. 33a , when no force is applied to the elastic layer 6,the elastic layer 6 is in its ‘at rest’ state and is planar, permittingfluid flow through the channel 8. When force is applied to the elasticlayer 6 in the region of the channel 8, the elastic layer 6 is deformedin the region of the channel 8, thereby affecting fluid flow in thechannel 8. In the example of FIG. 33b , the force applied to the elasticlayer 6 is sufficient to cause the elastic layer 6 to contact theopposing wall of the channel 8, thereby causing the channel 8 to close.Thus the application, or removal, of force to the elastic layer 6 in theregion of the channel 8 can be used to cause a portion of the channel 8to operate as a valve.

Although FIGS. 33a and 33b show a single channel that is operable as avalve, any desired number and arrangement of channels and/or chamberscan be provided in the microfluidic device, and force can be selectivelyapplied to deform any part of the elastic layer in order to controland/or otherwise manipulate fluid flow or fluid dynamics in the chambersor channels in any way that is desired.

It is a feature of the described embodiments that the microfluidicdevice is detachable from a control platform that can be used to applyforce to the elastic layer in order to control fluid flow within themicrofluidic device.

A control platform 10 is illustrated in FIG. 34. The control platformcomprises a microactuator mechanism 12 disposed on a face 14 of thecontrol platform 10. The microactuator mechanism is linked to acontroller 16 that is operable to control operation of the microactuatormechanism 12. The microactuator mechanism is operable to move amicromechanical element (indicated by dotted lines in FIG. 34). When theface 14 of the control platform is engaged with the elastic layer 6 ofthe microfluidic device 2, operation of the microactuator mechanism 12causes the micromechanical element to apply a force to, and thus deform,a corresponding portion of the elastic layer 6.

The controller is, for example, a general purpose computer programmedwith suitable control and interfacing software, or may be a suitablededicated hardware device, for example comprising one or more ASICs(application specific integrated circuits).

The part of the elastic layer 6 that is deformable by the microactuatormechanism 12 can be selected by aligning the microactuator mechanism 12with the selected part of the elastic layer 6. In the example of FIGS.33a and 33b , the microactuator mechanism 12 is aligned with the part ofthe elastic layer 6 that forms a wall of the channel 8 if it is desiredto open or close the channel 8. Operation of the microactuator mechanism12 then closes the channel 8. Subsequent deactivation, or removal, ofthe microactuator mechanism 12 relieves the elastic layer, returning theelastic layer to a planar form, and re-enabling fluid flow in thechannel 8.

The microactuator mechanism 12 can be any electromechanical orelectromagnetic device controllable by application of electrical currentor magnetic field to move a micromechanical element to deform theelastic layer. Any suitable, known electromechanical or electromagneticdevice may be used. Alternatively the microactuator mechanism maycomprise a heating or cooling device that is operable to deform theelastic layer by selectively heating or cooling parts of the elasticlayer. Alternatively, the microactuator mechanism may be operable todeform the elastic layer by applying fluid pressure, for example via apneumatic or vacuum system. The microactuator mechanism may be formed ofany suitable material, including (but not limited to) silicon, glass,ceramic, metal or polymer material.

In the embodiment of FIG. 34, the dimensions of the control platform 10match the dimensions of the microfluidic device 2. The position of themicroactuator mechanism 12 on the face of the control platform 10corresponds to the position on the microfluidic device 2 of that part ofthe elastic layer 6 forming the wall of the channel 8. Thus, if theedges of the control platform 10 are aligned with the edges of themicrofluidic device 2, as shown in FIG. 35, and the control platform 10and the device 2 are brought together the microactuator mechanism 12opposes that part of the elastic layer 6 forming the wall of the channel8, and operation of the microactuator mechanism 12 opens or closes thechannel 8. Thus, a simple technique for correctly aligning the controlplatform 10 and the microfluidic device 2 is provided. The rigid layer 4gives the device 2 a well-defined shape, more easily enabling precisealignment with the underlying control platform 10.

Once the control platform 10 and the microfluidic device 2 have beenaligned to a desired position and coupled together so that operation ofthe microactuator mechanism 12 causes deformation of the elastic layer6, they are fixed together using screws that pass through fixing holes(not shown) in the microfluidic device 2 and are screwed into threadedholes (not shown) in the control platform 10. Any other suitable fixingarrangement can be used, for example a clamp, nuts and bolts, orreleasable adhesive.

In the embodiment of FIGS. 34 and 35, the control platform 10 and themicrofluidic device 2 can be aligned merely by aligning their edges. Inalternative embodiments, further alignment features are provided. Forexample, a disposable microfluidic device 22 is shown in FIG. 36, whichis provided with alignment holes 24. The control platform 20 comprisesfour precisely positioned alignment pillars 26 located at its corners,two of which are shown in FIG. 36. The microfluidic device 22 can beprecisely aligned with the control platform 20 by inserting thealignment pillars 26 into the alignment holes 24. The disposablemicrofluidic device 22 can be easily plugged in to and thus correctlyaligned with the control platform 20.

Various alternative alignment features are provided in differentembodiments. For example, in some embodiments, various differentpositions of a microfluidic device on the control platform are used,depending on the operations to be performed on the microfluidic deviceand/or the type of microfluidic device to be coupled to the platform. Insome such embodiments, alignment holes are provided on both themicrofluidic device and the control platform, and different alignmentpositions can be selected by inserting pins between different pairs ofalignment holes. Although the use of alignment pillars or pins andalignment holes has been described, any type of male and femaleconnectors can be used to align the microfluidic device and the controlplatform. Alternatively or additionally, alignment marks are provided onthe control platform and the microfluidic device that are aligned whenthe control platform and the microfluidic device are in a correctposition.

In the embodiment of FIG. 34, force is applied by the control platformto a part of the elastic layer of the microfluidic device by movement ofa mechanical element driven by an electromechanical micro-actuatormechanism. As mentioned above, force can also be applied to deform theelastic layer by applying fluid pressure to the elastic layer, and anexample of an embodiment that uses such application of fluid pressure isillustrated in FIGS. 37a and 37b . A microfluidic device 30 is shown incross-section in FIG. 37a and comprises a fluid chamber 32 connected toa fluid channel, both formed within a rigid layer 36 of the microfluidicdevice 30. The elastic layer 6 forms a wall of the fluid chamber 32. Thefluid channel runs in a direction perpendicular to the plane of thefigure and is not shown in FIG. 37 a.

A control platform 40 that is coupleable to the microfluidic device 30for control of operation of the microfluidic device 30 is also shown inFIG. 37a . The control platform 40 comprises a fluid channel 42 that isconnectable to a gas supply 44. The gas supply 44 is linked to acontroller 46 that is operable to control the gas supply 44 to supplypressurised gas to the gas channel 42. An output 48 of the gas channelis provided in a face 50 of the control platform 40. An O-ring 52 isprovided that is disposed on the face 50 around the output 48 of the gaschannel. The controller 46 comprises at least one valve for controllingthe supply of gas from the gas supply 44 and a suitable general purposecomputer programmed with suitable control and interfacing software forcontrolling the at least one valve. The general purpose computer may bereplaced by a suitable dedicated hardware device, for example comprisingone or more ASICs (application specific integrated circuits).

In order to perform operations on the microfluidic device 30, thecontrol platform 40 and the microfluidic device are aligned and fixedtogether as shown in FIG. 37b . The O-ring 52 is compressed between theface 50 of the control platform 40 and the elastic layer 6, and forms anair-tight connection that seals a volume connecting the output 48 of thegas channel and the part of the elastic layer 6 that covers the chamber32.

In operation, pressurised gas is supplied by the gas supply 44 via thegas channel 42 to the sealed volume. The pressurised gas in the sealedvolume applies a force to the elastic layer 6 over an area B defined bythe O-ring. The pressurised gas causes the part of the elastic layer 6forming a wall of the chamber 34, and having an area A, to deform and tocause fluid to flow from the chamber 34 into the channel. The chamber 34and the channel form part of a microfluidic mixing device and, inoperation, the controller 46 causes pressure to be applied and releasedfrom the sealed volume repeatedly in order to repeatedly deform andrelax the elastic layer 6, thus contributing to a mixing of the fluid inthe mixing device.

In variants of the embodiment of FIGS. 37a and 37b a vacuum, orunder-pressure, rather than an over-pressure is applied to the elasticlayer (for example, by pumping the sealed volume defined by the O-ring).In such embodiments, the elastic layer in its normal state can be incontact with the opposing wall of the chamber or channel and theapplication of the vacuum, or under-pressure, causes the elastic layerto move away from the opposing wall, opening the chamber or channel.

It is a feature of the embodiment of FIGS. 37a and 37b that the area Aof the elastic layer that is deformable to cause operation of themicrofluidic mixer (or other types of microfluidic control components,in other embodiments) is smaller than the area B over which force isapplied to the elastic layer by the control platform 40. The use ofpneumatics or other fluid pressurisation techniques to apply force tothe elastic layer provides for greater tolerance in the alignment of thecontrol platform and the microfluidic device, when the area over whichforce is applied is greater than the area of the elastic layer that isto be deformed to perform microfludic operations. In such embodiments,it is sufficient that the larger area over which force is appliedcovers, or at least overlaps, the smaller area that is to be deformed.

The microfluidic devices can be attached and detached from the controlplatform, and operations perfomed on the microfluidic devices, withouthaving to change the set up of the control platform each time (themicrofluidic devices and control platform can have a plug and usedecoupled design). For example, for embodiments that use fluidpressurisation techniques, such as the embodiment of FIG. 37, the gassupply 44 and the controller 46 can remain connected to the controlplatform 50 whilst a series of microfluidic devices can be attached toand detached from the control platform 40. There is no need to reconnectgas or other inputs each time the microfluidic device on the controlplatform is changed.

The decoupled design means that it is relatively straightforward toperform measurements or operations on a series of microfluidic devices,using the same control components provided on the control platform. Asthe microfluidic devices have a simple structure, are relativelystraightforward to manufacture, and do not need to include complexelectromechanical devices, or sensors, they can be treated asdisposable, if desired.

In some cases a series of measurements can be performed by the samecontrol platform on microfluidic devices containing a series ofdifferent fluid samples or fluid samples under a series of differentconditions. In one example, a series of measurements or operations canbe performed on a series of different volumes of the same sample, byattaching a series of microfluidic devices in turn, each microfluidicdevice having a sample chamber of a different size. The embodiment ofFIG. 37, for example, is suitable for performing such a series ofmeasurements or operations, particularly if the area of the samplechamber in each case is smaller than the area over which the force isapplied by the control platform (the area contained by the O-ring inFIG. 37).

The embodiments described in relation to FIGS. 33 to 37 include amicrofluidic device having a single chamber or channel on whichoperations are performed by deforming the elastic layer, and a controlplatform having a single microactuator mechanism or other feature forapplying force to the elastic layer. In practice many different chambersor channels can be included on the same microfluidic device, each ofwhich can be used to perform microfluidic operations under control of asingle control platform having multiple microactuator mechanisms orother devices for applying force, or performing measurements or otheroperations.

An embodiment with multiple channels, and multiple locations on thecontrol platform that are used to apply force is illustrated in FIGS.38a to 38c , and comprises a microfluidic device 60 made of Polymer SU-8on a glass substrate, which contains three microfluidic chambers 61, 62,63 linked by a channel 64 having channel dimensions 200 μm (width) by 5cm (length) by 100 μm (thickness). The elastic layer 65 is formed ofbonded silicone film of thickness 80 μm. The control plate 66 is made ofa plastic material, PMMA, of dimensions 3 cm (width) by 5 cm (length) by1 cm (thickness), and contains three pneumatic components eachcomprising an O-ring (not shown) and a gas channel 67 a, 67 b, 67 c andoutput 68 a, 68 b, 68 c that are operable to apply force to the elasticlayer 65. The pressure or vacuum applied via the outputs 68 a, 68 b, 68c in operation causes deformation of the elastic layer 65, which canmanipulate the fluid in the microfluidic device.

The control platform can also include various components to performoperations on the fluids in the microfluidic devices. The decouplednature of the technology enables a high degree of flexibility in thecontrol of reactions compared to existing microfluidic devices, allowingincorporation of any active micro-components for reaction control and/ormonitoring into the control platform. This may include (but is notlimited to) the integration of microheaters, micromagnets, microdiodes(UV or other), and micro-optical or other detectors or sensors to thecontrol platform for use in any kind of applications, includingbiomedical applications.

This flexibility also extends to the fabrication of the microfluidicdevice. For example, a single type of microfluidic device may be builtto perform all types of reactions on multiple different controlplatforms, each built for a different function. Alternatively, a singlecontrol platform can be used to perform all types of reactions onmultiple different microfluidic devices (also referred to as chips),each built for a different function.

For some components, for example microheaters, micromagnets, and atleast some types of sensor or detector, it can be important to havedirect contact or at least a minimum distance between the component andthe microfluidic device, in order for the component to perform itsfunction correctly on the fluid within the microfluidic device.

Contact, or at least a sufficiently small gap, between components of thecontrol platform and the microfluidic device can be provided by mountingthe components on the control platform with springs, elastic cushioningmaterial or other biasing elements for ensuring that the componentsprotrude above the face of the control platform. That can beparticularly important for embodiments in which fluid pressure is usedto apply force, and in which an O-ring or other sealing mechanism isused to create a seal between the face of the control platform and themicrofluidic device, as O-rings or other sealing mechanisms are usuallyof non-negligible thickness and leave a gap between the control platformand the microfluidic device.

An example of such an embodiment is illustrated in cross-section in FIG.39, which shows a control platform 70 for use with a microfluidic device90. The microfluidic device is similar to that illustrated in FIGS. 37aand 37b , but includes a further chamber 92 connected to a further fluidchannel. The channel and the further channel run in a directionperpendicular to the plane of the figure and are not shown in FIG. 39.The control platform 70 comprises a pressurised gas channel 72 andoutput 74 for applying pressure to the elastic layer 6 when coupled tothe microfluidic device 30. An O-ring 76 is provided to form a sealbetween the elastic layer 6 and the face 78 of the control platform 70around the output 74. The control platform also includes a furthercomponent, in this case a microheater 80 that can be aligned with, andheat fluid in, the further chamber 92. The microheater 80 is mounted onsprings 82, 84. The springs 82, 84 bias the microheater 80 away from theface of the control platform 70. It can be seen from FIG. 39 that whenthe face of the control platform and the elastic layer of themicrofluidic device are not in contact, the microheater protrudes fromthe face of the control platform above the level of the O-ring 76.

When the face of the control platform and the elastic layer, or othersurface, of the microfluidic device are clamped or otherwise joinedtogether, the microheater component 80 is contacted by the elastic layer6 and is at least partially pushed into the body of the control platformuntil the elastic layer contacts and is sealed against the O-ring 76.Good contact is maintained between the microheater component 80 and theelastic layer 6 by the biasing effect of the springs 82, 84.

As has already been mentioned, the deforming of the elastic layer of themicrofluidic device in the region of one or more fluid channels orchambers can cause the fluid channels or chambers to operate asmicrofluidic control components, for example valves, mixers or pumps.

The operation of a fluid channel as a valve has already been describedin relation to FIG. 33. A further embodiment in which a fluid channel isoperated as a valve is illustrated in FIGS. 40a and 40b , which shows ina planar view a fluid flow channel 100 of dimensions 1 mm (width) by 5mm (length) by 100 μm (thickness) formed in a glass substrate, and thatcomprises a microvalve region 102. The glass substrate is covered withan elastic layer formed of SU-8 material, which forms a wall of thefluid flow channel 100. Fluid flow through the channel 100 in themicrofluidic device is controlled using the microvalve region 102. Fluidcan flow through the channel 100 when the microvalve region 102 of thechannel is open (the microactuator mechanism of the control platform isnot applied to the elastic layer, elastic layer is planar) as indicatedschematically in FIG. 40a . Fluid flow through the channel is stopped asindicated schematically in FIG. 40b when the microvalve region 102 ofthe channel 100 is closed (the microactuator mechanism is applied to theelastic layer in the microvalve region 102, elastic layer is deformed,blocking the channel).

A micropump component can be also be formed, and uses a similarmechanism to the microvalve, based upon the application of force todeform the elastic layer so that it is forced into a fluid flow channelor chamber in the rigid layer of the microfluidic device. However, in amicropump the deformation of the elastic layer may be such as to notcompletely close the channel or chamber and thus not to preclude fluidflow through the channel or channel. Instead the deformation of theelastic layer of the micropump component forces the fluid to flowthrough or out of the channel in one or more directions.

FIGS. 41a and 41b illustrate schematically the operation of a micropumparrangement 110 implemented using a decoupled two-layer microfluidicdevice. The micropump arrangement 110 represented schematically inplanar view in FIGS. 41a and 41b comprises a microfluidic devicecomprising a fluid flow channel 112 of dimensions 1 mm (width) by 15 mm(length) by 100 μm (thickness) formed in a glass substrate, and havingan opening 111, 113 at each end. The fluid flow channel 112 comprisestwo microvalve regions 114, 116 flanking a micropump region 118 in whichthe fluid flow channel 112 widens to form a circular microchamber ofdimensions 5 mm (diameter) and 100 μm (depth). The glass substrate iscovered with an elastic layer in the form of a membrane of SU-8material, that forms a wall of the fluid flow channel. The microfluidicdevice illustrated in FIGS. 38a to 38c has a geometry that is suitablefor use in the micropump arrangement of FIGS. 41a and 41 b.

The microfluidic device is aligned with and coupled to a controlplatform, such that microactuator mechanisms of the control platform arealigned with and individually operable to deform the elastic membrane atthe two microvalve regions 114, 116 and at the micropump region 118.

In order to perform a pumping operation, the control platform repeatedlyoperates the microactuator mechanisms in a predetermined sequence. Inthe first stage of sequence, the microactuator mechanism adjacent to thefirst microvalve 114 is activated to close the first microvalve 114,whereas the second microvalve 116 remains open. The microactuatormechanism adjacent to the micropump region 118 is then activated toforce fluid out of the microchamber. As the first microvalve 114 isclosed, the fluid is forced along the channel 112 towards and throughthe second valve 116.

In the next stage of the sequence, the microactuator mechanism adjacentto the first microvalve 114 is de-activated to open the first microvalve114, and the microactuator mechanism adjacent to the second valve isactivated to close the second microvalve 116 (the microactuatormechanism adjacent to the microchamber remains activated during thoseoperations).

The microactuator mechanism adjacent to the microchamber is thendeactivated, releasing the elastic membrane adjacent at that locationand pulling fluid through the opening 111 to the channel 112, andtowards and through the first microvalve 114 and the microchamber, inthe direction of the (now-closed) second microvalve 116.

The sequence is then repeated, to pump fluid through the channel 112 ina controlled fashion. Performing the sequence of operations in reversepumps fluid through the channel 112 in the opposite direction.

The size of the microchamber and/or the fluid flow channel 112 can bevaried in order to vary the pumping rate or other properties of thepump. For example, in variants of the embodiment of FIGS. 41a and 41b ,the diameter of the microchamber varies between 0.05 mm and 5 mm indiameter.

It has been found that the pumping rate of the pump can also be variedby varying the frequency at which the sequence of stages is repeated(and thus the frequency at which the elastic membrane is deformed andallowed to relax). A graph of pumping rate as a function of frequency ofoperation (equal to the frequency of activation of the microactuatormechanism adjacent to the microchamber of the micropump in this case) isprovided in FIG. 42. The flow rate is proportional to frequency untilthe frequency reaches the resonant frequency of the membrane. When theactuation frequency greater than the resonant frequency of the elasticmembrane in the region of the microchamber, the maximum amplitude ofdeformation of the elastic membrane is not fully achieved. Therefore,the pumping volume is reduced upon further increase of the actuationfrequency.

The repeated deformation of the elastic layer can also be used toprovide mixing effects. In one example, the micropump of FIG. 41 can beoperated as a mixer. The opening 111 of the fluid flow channel 112 isconnected to one source of fluid, and the other opening 113 is connectedto another source of fluid, and the fluids from the two sources areallowed to pass to the microchamber. The microactuator mechanisms arethen operated to close the microvalves 114, 116. With the microvalves114, 116 closed, the microactuator mechanism adjacent to themicrochamber is then repeatedly activated at a frequency (for example,greater than 100 Hz) much higher than the resonant frequency of thatpart of the elastic membrane forming a wall of the microchamber (forexample, the resonant frequency in the embodiment of FIG. 41 is around10 Hz). By operating at such a frequency, the elastic layer is deformedwith an amplitude (for example 5 microns) that may be smaller than theamplitude obtainable if operating at a frequency lower than the naturalfrequency, but that is sufficient to induce mechanical disturbance inthe fluids to mix the fluids inside the microchamber. The fluids may beboth be liquids, or at least one of the fluids may be a gas.

In another arrangement, a microchamber is used as a mixing chamber byrepeatedly deforming the elastic layer forming a wall of the mixingchamber, as described in the preceding paragraph, but instead of thefluid being constrained to the mixing chamber by the closure of valveson both sides of the mixing chamber (for example microvalves 114, 116described in the preceding paragraph) the microchamber is connected toan open fluid flow channel on each side and the fluid is mixed as itflows through the microchamber.

In another arrangement, each end 111, 113 of the fluid flow channel isconnected to a respective microchamber. In that arrangement, themicropump is operated to alternately pump the fluids to be mixed betweenthe microchambers in one direction and then in the reverse direction. Ithas been found that repeating the pumping operation in one direction andin the reverse direction more than once is sufficient to mix two fluids.

The microfluidic control components described in relation to FIGS. 40and 41 may also be implemented in integrated microfluidic structuresthat comprise both fluid channels and/or chambers and components formanipulating the fluid in the channels and/or chambers. The microfluidiccontrol components do not have to implemented in a decoupled structuresuch as those described in relation to FIGS. 33 to 39, in which acontrol platform is coupleable and decoupleable from a microfluidicdevice.

Various materials for use as the elastic layer have been described, butthe elastic layer is not limited to being formed of such materials. Anysuitable material can be used for the elastic layer, for examplesilicone, polyurethane elastomer, butyl rubber, nitrile rubber, ethyleneacrylic elastomer, ethylene propylene rubber, natural rubber, styrenecontaining block copolymer elastomers, santoprene elastomer andpolychroroprene elastomer. The elastic layer can be of any suitablethickness, and the most appropriate thickness may depend on themicrofludic operations to be performed and on the size and arrangementof the microfluidic chambers and channels. For the embodimentsillustrated in FIGS. 33 to 42, it has been found that it is desirablefor the elastic layer to have a thickness less than or equal to 250 μm.

The embodiments described in relation to FIGS. 33 to 39 have included arigid layer that is substantially rigid in its entirety. In alternativeembodiments, the rigid layer comprises a substantially rigid frameworkand flexible or other material attached to the substantially rigidframework.

The microfluidic control platform can be formed of any suitablematerial, and is usually formed of a rigid material, for example glass,plastic, polymer or ceramic.

The microfluidic systems can be used for manipulation of or operationson microfluidic amounts of fluids, either gases or liquids, for anypurpose. Any type of sample may be manipulated or operated on using thesystems. Examples of samples include but are not limited to:—particulatematter including nano-particles, quantum dots, polymer or magneticbeads; organisms; organs; tissues (such as tumour biopsies and bloodvessels); cell samples, samples of cell derived parts or substances, anycells or eukaryotic or prokaryotic origin such as primary cell cultures,stem cells and cell lines, and including animal, plant, yeast andbacterial cultures. The samples may be samples for a biological orbiochemical assay such as, for example, blood, urine, saliva, cellderived part or substance (such as proteins, genes, genomes, DNA, RNA,organelles such as mitochondria or ribosome, or cell or organellemembranes).

Certain embodiments may eliminate the need to integrate microactuatorcomponents onto a microfluidic device, making device fabrication andinvestigation significantly less complex than existing systems,therefore lowering manufacturing costs, increasing the potential forhigh value manufacture, and also contributing to the disposability ofmicrofluidic devices.

Certain embodiments open the possibility of modular microfluidic devicefabrication, giving the potential to easily change and assemble custommicrofluidic systems for different applications, as determined by an enduser.

Magnetic means can be used for the trapping and/or mixing of nucleicacid sequences (parts) and/or oligonucleotide linker sequences (oligos)which are bound to magnetic beads in the microfluidic device. Magneticmeans can also be used for the purification of nucleic acid sequencesimmediately prior to step (iv) of the method of the invention, asdescribed herein.

Magnetic particles or beads can be chemically treated to make thembiologically active, which causes them to bind to other biologicalcomponents, such as nucleic acid sequences or oligonucleotide linkersequences, present in a reaction mixture. In one embodiment, wheremagnetic particles or beads are used for the purification of nucleicacid sequences, the magnetic particles or beads bind to partpurification oligos, as defined herein.

When the magnetic particles bind to the desired biological components,the magnetic particles with biological components attached can be pumpedthrough the microfluidic device, for example from the input chamber tothe reaction chamber, as for a fluid. The magnetic particles can then beretained in a particular chamber, for example the reaction chamber,using a magnetic field, generally generated by a hard magnet. Themagnetic particles can then be removed from a particular chamber, forexample the reaction chamber, by removing the magnetic field and thenpumping new fluid into that chamber to wash the beads into anotherchamber, for example an output chamber. In one embodiment, a magneticfield is produced manually by holding a magnet close to a particularreaction chamber and then removed by taking the magnet away from thereaction chamber.

A magnet can also be used to mix the magnetic particles with biologicalcomponents attached. For example, this can be done manually by holding amagnet close to a particular chamber and moving the magnet, for examplein a circular motion.

Magnetic particles can therefore be used both to purify and mix productsin a microfluidic device. In one embodiment of the method of theinvention therefore, the nucleic acid sequences are bound to magneticbeads and said magnetic beads are trapped and/or mixed in any one ofsaid chambers using magnetic means.

The magnetic beads used are typically superparamagnetic nanoparticles,such as those made of iron oxide. The magnetic beads are of a sizesuitable for use in a microfluidic device as described herein, and sothe diameter of such beads will be in the μm range, for example from 0.5μm to 3 μm, from 1 μm to 2 μm, typically around 1 μm.

In one embodiment, the magnetic beads are coated with or covalentlyattached to streptavidin. In this embodiment, the magnetic beads bind tobiotin which can be coupled to a nucleic acid sequence, oligonucleotidelinker sequence or other oligo, such as a part purification oligo asdefined herein. Streptavidin magnetic beads are available, for example,from New England Biolabs, MA.

Trapping and mixing of magnetic particles can be carried out using hardmagnets such as neodymium (NdFeB) magnets, optionally including softiron parts. In one embodiment, the magnetic means comprises rotatingmachined hard magnets or stacks of hard magnets with machined soft ironparts.

In this embodiment, hard magnets can be produced with very specificfeatures such as those shown in FIG. 18 which then, by rotating themagnet, induce both trapping and mixing of the magnetic particles. Thefeatures can be cut, for example, by machining from one end using powderblasting, or a grinding process using diamond tools.

Alternatively, hard magnets can be connected to soft iron parts, onwhich the desired features (for example as shown in FIG. 19) have beenproduced, typically by machining using standard machining/milling tools.Then, the stack of hard magnet/soft iron part is rotated to induce boththe trapping and mixing effect

In this embodiment, commercially available amorphous magnetic foils (forexample Iron or Cobalt based magnetic alloys), with very high magneticpermeabilities (for example with μ_(r) from 10⁵ to 10⁶), can be used tocover the machined and exposed areas of the hard magnet (see FIG. 20),to screen the magnetic field emanating from these areas. This fieldmight actually overlap with the field generated from the upperstructures, and tends to homogenize the applied magnetic field on thereaction chamber. This forces the particles to concentrate around thecentre of the chamber minimizing their rotation and mixing effect. Thusthe use of an amorphous magnetic foil screens the magnetic field comingfrom these areas which might overlap with the active field and wouldotherwise reduce the mixing efficiency. In one embodiment, the magnethas a teardrop shape, as shown in FIG. 20.

Alternatively, another magnet can be used to magnetize the magneticparticles, with a weaker magnetic field strength in order to allow forthe driving magnetic field to act more efficiently on the particles. Themagnetic field can be applied either from the bottom of the reactionchamber, by direct application or through a soft iron material, or fromthe sides of the chips by using large hard magnets which would allow ahomogeneous magnetic field distribution around the reaction chamber.

In the case of mixing the magnetic particles in the reaction chambercovered by a flexible membrane, being sealed and having an airconnection, the mixing can be further enhanced by pulsing the airconnection inducing a slight deflection of the membrane, which forcesthe magnetic particles to interact even more with the differentsubstances present in the chamber. This process is schematicallyrepresented in FIG. 21.

Trapping and mixing of magnetic particles can alternatively be carriedout using magnetic coils, for example magnetic coils arrayed and stackedon a planar printed circuit board (PCB) platform, or manufactured bywinding enamelled copper wires. In this embodiment, the magnetic meanscomprises magnetic coils to which a magnetic field is applied.

In this embodiment, different geometries of the magnetic coils can beused, such as rectangular or semi-circle shaped coils, arrayed andstacked together. FIG. 22 shows different geometries designed for thePCB board (FIG. 22a ) and for the winding of enamelled wires (FIGS. 22band 22c ).

A simple configuration consists of a stack of two layers of coils asshown on FIG. 22. An alternative configuration is to use four layers ofcoils, two of which define magnetic channels for the magnetic particlesto be confined to, while driving them for the other set of two.

The principle behind trapping the magnetic particles relies on thecombination of two components of magnetic fields. The first component isprovided by either one hard magnet, when applied from the bottom of thechip through a soft iron part (FIG. 23a ), or a stack of several hardmagnets when applied from the sides of the chip (FIG. 23b ). Both topand side views of the two configurations are shown on FIGS. 23a and 23b. The second component of the magnetic field applied on the magneticparticles is provided by the magnetic coils. The magnetic field gradientinduced by the time varying applied electrical current crossing thesecoils will be enough to trap them.

In a similar manner, mixing of the magnetic particles can be performedby driving the particles in a rotational movement in the reactionchamber. This can be realized by passing a periodic square currentthrough the coils in a sequence that allows dragging the particles fromone centre of coil to the nearest one, with speeds around a fewmillimeters per second, until the particles make a 360° turn in thereaction chamber.

Depending on the rotation speed of the magnetic particles in thereaction chamber, this approach can be used for magnetic trapping andseparation of magnetic particles; as a magnetic stirrer (at relativelylow speeds up to a few hundred RPMs) to stir and mix different fluidsand products in the reaction chamber; or as a magnetic mixer (atrelatively high speeds more than 300 RPMs) to break the bonds in theaggregates of magnetic particles and mix them with the different liquidsand reagents involved in the processes.

The method of the invention can also be used for the preparation of acombinatorial library of nucleic acid sequences.

According to a second aspect, the present invention therefore provides amethod for the preparation of a library of polynucleic acid sequences,the method comprising simultaneously producing a plurality of differentpolynucleic acid sequences using the method of the first aspect of theinvention.

In one embodiment of the second aspect of the invention, the method forthe preparation of a library of polynucleic acid sequences comprisessimultaneously carrying out the method of the first aspect of theinvention, i.e.

-   -   (i) providing a first nucleic acid sequence N1 which has an        oligonucleotide linker sequence L1^(3′) at the 3′-end of the        nucleic acid sequence;    -   (ii) providing a second nucleic acid sequence N2 which        optionally has an oligonucleotide linker sequence L2^(3′) at the        3′-end of the nucleic acid sequence and which has an        oligonucleotide linker sequence L2^(5′) at the 5′-end of the        nucleic acid sequence,        -   wherein the 5′-end linker sequence L2^(5′) of nucleic acid            sequence N2 is complementary to the 3′-end linker sequence            L1^(3′) of nucleic acid sequence N1;    -   (iii) optionally providing one or more additional nucleic acid        sequences N, wherein nucleic acid sequence N2 has an        oligonucleotide linker sequence L2^(3′) at the 3′-end of the        nucleic acid sequence, and wherein said one or more additional        nucleic acid sequences N comprises a terminal additional nucleic        acid sequence NZ, and wherein each additional nucleic acid        sequence N has an oligonucleotide linker sequence at its 3′-end,        wherein said terminal additional nucleic acid sequence NZ        optionally lacks an oligonucleotide linker sequence at its        3′-end and wherein each additional nucleic acid sequence N has        an oligonucleotide linker sequence at its 5′-end,        -   wherein for the first additional nucleic acid sequence N3            the 5′-end linker sequence L3^(5′) is complementary to the            3′-end linker sequence L2^(3′) of nucleic acid sequence N2            and for each second and subsequent additional nucleic acid            sequence N the 5′-end linker sequence is complementary to            the 3′-end linker sequence of the respective preceding            additional nucleic acid sequence;        -   and    -   (iv) ligating said nucleic acid sequences to form said        polynucleic acid sequence;        wherein at least step (iv) is carried out on a microfluidic        device;        a plurality of times with different combinations of nucleic acid        sequences N, thereby producing a plurality of different        polynucleic acid sequences.

In this embodiment, the method of the first aspect of the invention iscarried out a plurality of times simultaneously, and the output of thismethod is “n” distinct samples with “n” defined assemblies, i.e. thenumber of samples is equivalent to the number of assemblies.

In another embodiment of the second aspect of the invention, the methodfor the preparation of a library of polynucleic acid sequences comprisescarrying out the method of the first aspect of the invention once, butcarrying out the method with a mix of nucleic acid sequences for one oreach of the nucleic acid sequence N1, N2 etc.

For example, for a 3-part assembly, the method of the first aspect ofthe invention comprises the following steps:

-   -   (i) providing a first nucleic acid sequence N1 which has an        oligonucleotide linker sequence L1^(3′) at the 3′-end of the        nucleic acid sequence;    -   (ii) providing a second nucleic acid sequence N2 which has an        oligonucleotide linker sequence L2^(3′) at the 3′-end of the        nucleic acid sequence and which has an oligonucleotide linker        sequence L2^(5′) at the 5′-end of the nucleic acid sequence,        -   wherein the 5′-end linker sequence L2^(5′) of nucleic acid            sequence N2 is complementary to the 3′-end linker sequence            L1^(3′) of nucleic acid sequence N1;    -   (iii) providing a third nucleic acid sequence N3 which has an        oligonucleotide linker sequence L3^(5′) at the 5′-end of the        nucleic acid sequence,        -   wherein the 5′-end linker sequence L3^(5′) of nucleic acid            sequence N3 is complementary to the 3′-end linker sequence            L2^(3′) of nucleic acid sequence N2;        -   and    -   (iv) ligating said nucleic acid sequences to form said        polynucleic acid sequence;        wherein at least step (iv) is carried out on a microfluidic        device.

In one embodiment of the second aspect of the invention, the method ofthe first aspect of the invention can be carried out using a randommixture of a number of different nucleic acid sequences, for example 3different nucleic acid sequences to replace N1: N1a, N1b, and N1c. Eachof the different nucleic acid sequences has the same oligonucleotidelinker sequences L1^(5′) and L1^(3′). Each of the nucleic acid sequencesN2 and N3 can also be replaced by a set of similar variants, e.g. N2a,N2b and N3a, N3b, N3c, N3d. All variants within each set have the sameoligonucleotide linker sequence so that any assembly that includes onepart from the N1 set, one part from the N2 set, and one part from the N3set could form. For example, N1b, N2a, N3c is one possible assembly.

If the method of the first embodiment of the invention is carried outusing 3 variants for nucleic acid sequence N1, 2 variants for nucleicacid sequence N2 and 4 variants for nucleic acid sequence N3, it can beseen that the resulting assembly will produce a library containing arandom set of assemblies. In this example, there would be 24 possibleassemblies that are generated in the combinatorial library.

In this embodiment, the output of this method is thus one randomizedsample containing many different assemblies. This method can be used tocreate multiple different assemblies in a single reaction by varying thenucleic acid sequences at each position of the assembly as required.

The present invention also extends to the microfluidic device itself.

According to a third aspect, the present invention therefore provides amicrofluidic device comprising at least one input chamber, at least onestorage chamber, at least one reaction chamber and at least one outputchamber and wherein each of said at least one input chamber, at leastone storage chamber and at least one output chamber is linked by aseparate fluid channel to said at least one reaction chamber. In oneembodiment, the reaction chamber is substantially elliptical. In oneembodiment, the microfluidic device comprises two input chambers, onestorage chamber, one reaction chamber and two output chambers.

According to a fourth aspect, the present invention therefore provides amicrofluidic device comprising at least two input chambers, at least oneauxiliary chamber and at least one output chamber, wherein each of saidat least two input chambers and said at least one auxiliary chamber islinked by a central fluid channel to said at least one output chamber.In one embodiment, one or more of the input chambers is linked to thecentral fluid channel by a further fluid channel. In one embodiment, oneor more of the auxiliary chambers is linked to the central fluid channelby a further fluid channel. In one embodiment, the microfluidic devicecomprises two auxiliary chambers and two output chambers.

According to a fifth aspect, the present invention therefore provides amicrofluidic device comprising at least one input chamber, at least oneauxiliary chamber, at least one reaction chamber, at least one wastechamber and at least one output chamber, wherein said at least one inputchamber is linked by a fluid channel to said at least one reactionchamber, said at least one reaction chamber is linked by a fluid channelto said at least one output chamber, said at least one auxiliary chamberis linked by a fluid channel to said at least one waste chamber, andwherein the fluid channel linking said at least one auxiliary chamber tosaid at least one waste chamber intersects the fluid channel linkingsaid at least one input chamber to said at least one reaction chamber.In one embodiment, the number of input chambers is equal to the numberof reaction chambers and the number of output chambers. In oneembodiment, the microfluidic device comprises one auxiliary chamber andone waste chamber. In one embodiment, an additional fluid channelbranches from the fluid channel that links one or more of said inputchambers to one or more of said reaction chambers and joins the fluidchannel that links one or more of said reaction chambers to one or moreof said output chambers.

In one embodiment of the third, fourth or fifth aspect of the invention,one or more of the fluid channels has one or more valves. In oneembodiment, one or more of the fluid channels has two valves and whereina pump chamber is located between said two valves.

In one embodiment, the chambers and the fluid channels of a microfluidicdevice according to the third, fourth or fifth aspect of the inventionare located between a rigid layer and an elastic layer and themicrofluidic device is configured so that deformation of the elasticlayer manipulates fluid if present in said chambers or said fluidchannels.

In one embodiment, the microfluidic device according to the third,fourth or fifth aspect of the invention is part of a microfluidic systemfurther comprising a control platform comprising means for deforming theelastic layer thereby to manipulate fluid in the at least one fluidchamber or channel.

Other features of the microfluidic device according to the third, fourthand fifth aspects of the invention are as described in relation to thefirst aspect of the invention

According to a sixth aspect, the present invention provides a method fordesigning nucleic acid sequences suitable for use in a method accordingto the first aspect of the invention, comprising:

-   -   (i) analysing a nucleic acid sequence;    -   (ii) generating oligonucleotide linker sets from each nucleic        acid sequence; and    -   (iii) checking each oligonucleotide linker set to identify        conflicting linker/part overhangs, dimerization, complement        binding and/or a linker-part binding region.

The input for this method is a nucleic acid sequence (or “part”).Typically, the nucleic acid sequence is in a database. In step (i) ofthe method of this aspect of the invention, the nucleic acid sequence isanalysed. In step (ii), oligonucleotide linker sets for use in themethod of the invention are generated for each nucleic acid sequence. By“oligonucleotide linker sets” is meant a pair of oligonucleotide linkersequences that bind to the 3′ and 5′ end of a particular nucleic acidsequence. In step (iii), each oligonucleotide linker set is checked toidentify sequences that would interfere with the ability of theoligonucleotide linkers to be used in the method of the invention. Suchsequences include conflicting linker/part overhangs, dimerization,complement binding and/or a linker-part binding region. The methodoptionally includes a further step (iv), in which if any such sequencesare identified, the oligonucleotide linker sets are altered to removeany such sequences.

This aspect of the invention allows component parts to be designed sothat they will be amenable to construction via the part-linker DNAassembly technology.

The method of the sixth aspect of the invention is illustrated in FIG.29.

According to a seventh aspect, the present invention provides a methodfor planning the assembly of a polynucleic acid sequence from aplurality of nucleic acid sequences to be carried out by a methodaccording to the first aspect of the invention, comprising:

-   -   (i) analysing a plurality of assemblies of a polynucleic acid        sequence, said plurality of assemblies comprising different        combinations of nucleic acid sequences and oligonucleotide        linker sequences;    -   (ii) checking each of said plurality of assemblies of a        polynucleic acid sequence to identify repeat parts, repeat        termini and/or dimerization events; and    -   (iv) if repeat parts, repeat termini and/or dimerization events        are identified, either correcting said assembly or warning the        user that assembly correction is not possible.

This aspect of the invention allows the determination of whichassemblies to construct in parallel and how to share sub-componentsoptimally across assemblies.

The method of the seventh aspect of the invention is illustrated in FIG.30. Exemplary inputs and outputs are shown in FIG. 31.

According to a eighth aspect, the present invention provides a systemcomprising means for carrying out the method according to the sixth orseventh aspect of the invention.

According to a ninth aspect, the present invention provides a computerprogram which, when run on a computer, implements the method accordingto the sixth or seventh aspect of the invention.

According to a tenth aspect, the present invention provides a computerreadable medium or carrier signal encoding a computer program accordingto the ninth aspect of the invention.

FIG. 32 shows how the bioinformatics aspects of the inventioninterrelate. The different aspects shown in FIG. 32 are as follows:

The method of the sixth aspect of the invention is referred to in FIG.32 as the Part Designer. The Part Designer queries the part database fora part sequence. Oligonucleotide linker sets are generated from the partsequence subject to the constraints in Tech Options as set out below.These constraints include the Tm of the linker-part binding region,sequence of the linker overhang, etc. If all constraints cannot besatisfied, the Part Designer reviews and modifies the failedoligonucleotide linker sets and returns the best set of oligonucleotidelinkers according to priorities outlined in Tech Options. The user iswarned of potential dimerization events, low Tm of complementarystrands, restriction sites present in the part sequence, etc.

Pathway Generator: The user may query the part database for individualparts or sets of attributes. Part database attributes may include thetype of the part (promoter, regulator, cassette, etc.), organism, startcodon, etc. The user then inserts a list of parts into the desiredposition on the pathway. Upon submission, the Pathway Generator createsa list of all possible assemblies.

The method of the seventh aspect of the invention is referred to in FIG.32 as the Assembly Planner. The user inputs a list of assemblies by handor from the Pathway Generator. The Assembly Planner checks each assemblyfor repeat parts, repeat termini, dimerization events, etc. If found,the Assembly Planner attempts to correct the assembly by tryingalternate oligonucleotide linker sets. If the Assembly Planner cannotcorrect the assembly, the user is warned. The user has the option ofoutputting an optimized assembly plan.

Tech Options: This module is a centralized repository of control optionsfor the Part Designer, Pathway Generator and Assembly Planner. Itincludes both design parameters and hardware limitations. Tech Optionscan be input by the user.

Part Database: The Part Database stores information about parts andoligonucleotide linker sets. The user may add parts and oligonucleotidelinker sets (through Part Designer) to the database.

Also provided herein is a method or microfluidic device substantially asdescribed herein with reference to the accompanying drawings.

In another aspect, the present invention provides a method of mixingand/or trapping carried out on a microfluidic device, said methodcomprising using magnetic means to mix and/or trap magnetic beads insaid microfluidic device. In one embodiment, the magnetic meanscomprises machined hard magnets or stacks of hard magnets with machinedsoft iron parts. In another embodiment, the magnetic means comprisesmagnetic coils to which a magnetic field is applied.

One embodiment of the invention is shown in Example 1. Example 1demonstrates a 2-part assembly using the plasmid pSB1C3 and DNA encodingeither GFP or RFP. Prior to carrying out the method of the invention,the parts are digested with the restriction enzyme EarI to produce thenecessary overhangs. A successful assembly of pSB1C3.GFP produces greencells and a successful assembly of pSB1C3.RFP produces red cells. Theparts used in Example 1 are shown in schematic form in FIG. 9.

Accordingly, in one embodiment the present invention provides a methodaccording to the first aspect of the invention, wherein the method is asdescribed in Example 1. In another embodiment the present inventionprovides a method according to the first aspect of the invention,wherein the method is as described in Example 3. In yet anotherembodiment the present invention provides a method according to thefirst aspect of the invention, wherein the method is as described inExample 4.

Preferred features of the second and subsequent aspects of the inventionare as described for the first aspect mutatis mutandis.

BRIEF DESCRIPTION OF THE FIGURES

The invention will now be further described by way of reference to thefollowing Examples and Figures which are provided for the purposes ofillustration only and are not to be construed as limiting on theinvention. Reference is made to a number of Figures, in which:

FIG. 1 is a schematic diagram of the method of the present invention. Inphase 1, the parts and linkers are prepared. In phase 2, parts areligated to appropriate linkers based on the desired pathway assemblies.In phase 3, all parts are ligated together. In this example, there are 3parts being assembled: part A, part B and the plasmid backbone.Depending on the ligation method used, the assembly may leave a standardscar sequence between the parts (e.g. 3 bp).

FIG. 2 is a schematic diagram of the part preparation phase of oneembodiment of the invention. Parts are prepared to have overhangs andare stored with a set of oligos associated with the part. The overhangat the 3′-end of Part A (truncated) is a standard 3 bp sequence commonto all parts in a library. The biotinylated oligo 5_(A) can be used forpurifying the part. The biotin is represented by the circle. Oligos3_(A) and 4_(A) are stored for use during the assembly process.

FIG. 3 is a schematic diagram of the part-linker fusion phase of oneembodiment of the invention. In the part-linker fusion phase, part A isligated with oligos for the next part B.

FIG. 4 is a schematic diagram of the pathway assembly phase of oneembodiment of the invention. In the pathway assembly phase, part-linkerfusions are ligated together.

FIG. 5 is a schematic diagram of the purification of the final assemblyin one embodiment of the invention. The final assembly can be purifiedvia biotinylated oligos (5_(A) and 4_(D)). The biotin is represented bythe circle.

FIG. 6 is a schematic diagram of a part-linker DNA assembly scheme usingpartially double-stranded oligonucleotide linkers.

FIG. 7 is a schematic diagram of a part-linker DNA assembly scheme usingpartially double-stranded oligonucleotide linkers and truncated parts.

FIG. 8 shows the expected flanking sequences (overhangs) on partsfollowing digest with (A) EcoR1/Spe1 and (B) SapI/EarI. It can be seenthat the parts prepared using EcoR1/Spe1 have standard 4-bp overhangs,whilst the parts prepared using SapI/EarI have standard 3-bp overhangs.

FIG. 9 is a schematic diagram of the protocol used for 2-part assembliesin Example 1.

FIG. 10 shows in more detail the protocol used for 2-part assemblies inExample 1. The letters in the last column of FIG. 10 refer to chipcycles shown in FIG. 28.

FIG. 11 shows a purification approach that can be used in thepart/linker pair purification step.

FIG. 12 shows the 3D layout of the microfluidic device used inExample 1. The two input chambers have parallel fluid channels linkingthem to the central elliptical reaction chamber. The fluid channels ofthe two input chambers have two valves and a pump chamber. The storagechamber has a fluid channel linking it to the central ellipticalreaction chamber. The fluid channel of the storage chamber also has twovalves and a pump chamber. The two output chambers are linked by a fluidchannel to the central elliptical reaction chamber. Control of thedevice is provided by 12 air channels, one temperature control and onemagnetic part.

FIG. 13 shows the dimensions of the microfluidic device used in Example1.

FIG. 14A shows the layout of another microfluidic device for use in thepresent invention. FIG. 14B shows the layout of asymmetric channels inthis microfluidic device. FIG. 14C shows nozzled tip channels in thismicrofluidic device.

FIG. 15 shows the layout of another microfluidic device for use in thepresent invention.

FIG. 16: Packaging of microfluidic membrane using laser cutting and onelayer of double-sided adhesive tape. The steps are as follows: 1. 3MAdhesive transfer tape 467 MP applied on the PMMA block. 2. Load thePMMA block into the Epilog tool. Load the CAD file and set theparameters on Corel Draw. 3. Laser rastering and vector cutting. Rasterparameters PMMA+tape: 25/100. Vector cutting PMMA+tape: 5/100/5000. 4.Peel the protective layer 5. Apply the membrane on the adhesive layerand deframe it. 6. Chip ready with an excellent bonding

FIG. 17: Cut-through packaging technique using two layers ofdouble-sided adhesive layers. The steps are as follows: 1. Laser cuttingof the 4 mm PMMA sheet, Microfluidic access, and edges of the chip. 2.Laser cut-through of the microfluidic channels in a 0.125 mm layer ofPMMA covered on both faces with 50 μm adhesive layer and protectivecover. 3. Release protective cover, align and bond the channels (2) ontothe PMMA block (1). Release the second protective cover and place themembrane.

FIG. 18 shows schematic views of the machining of hard magnets using (a)powder blasting and (b) milling or grinding using diamond coated tools;(c) shows some examples of possible machined structures on hard magnets.

FIG. 19 shows the use of machined soft iron parts to focalise themagnetic field applied in order induce the same effect as for themachined magnets

FIG. 20 shows the use of amorphous magnetic materials to cover theexposed or machined areas on the hard magnet.

FIG. 21 shows a schematic view showing how the slight vibrations of themembrane can help during the mixing process.

FIG. 22: Schematic views showing the layouts for the magnetic coils for(a) circular and rectangular geometries on PCB board, (b) rectangularand (c) semi-circular geometries for winding enamelled copper wire.

FIG. 23: Schematic top and side views of the magnetic coil based systemfor separation and mixing of magnetic particles; (a) shows aconfiguration for which the magnetizing field is applied from under thechip, where the coils are applied, through a machined soft iron part,and (b) shows a configuration in which the magnetizing magnetic field isgenerated by a stack of hard magnets applied on opposite sides of thechip.

FIG. 24: Isometric left and right views of the heating/cooling andelectromagnet sub-assembly.

FIG. 25: Isometric view of the mechanical assembly for a control unitfor a microfluidic device as described herein, which also includes theheating/cooling and electromagnet sub-assembly.

FIG. 26 is a diagram of the microfluidic functions and the differentbiological components involved in the first purification step of theplasmid pSB1C3 as described in Example 1.

FIG. 27 demonstrates an example of how to operate a pump to flow liquidfrom one of the chambers into another chamber in a microfluidic deviceas described herein.

FIG. 28 summarises a number of different fluidic steps that were used inExample 1.

FIG. 29 is a flowchart of the method of the sixth aspect of theinvention.

FIG. 30 is a flowchart of the method of the seventh aspect of theinvention.

FIG. 31 shows exemplary inputs and outputs of the seventh aspect of theinvention.

FIG. 32 is a schematic of the bioinformatics aspects of the invention.

FIGS. 33a and 33b are schematic illustrations of a microfluidic deviceaccording to one embodiment.

FIG. 34 is a schematic illustration of a control platform.

FIG. 35 is a schematic illustration, in perspective view, of themicrofluidic device and the control platform.

FIG. 36 is a schematic illustration of a microfluidic device and acontrol platform, which include alignment holes and pillars.

FIGS. 37a and 37b are schematic illustrations a microfluidic device anda control platform in a further embodiment.

FIGS. 38a to 38c are schematic illustrations of a microfluidic deviceand a control platform according to a further embodiment.

FIG. 39 is a schematic illustration of a microfluidic device and acontrol platform according to another embodiment.

FIGS. 40a and 40b are schematic illustrations of a microfluidic valve.

FIGS. 41a and 41b are schematic illustrations of a microfluidic pump ormixer.

FIG. 42 is a graph of pumping rate as a function of actuator operationfrequency for the microfluidic pump of FIGS. 40a and 40 b.

FIG. 43: (A) Chemical structure of the indole-derivative, Violacein. (B)The biosynthesis of violacein from the precursor, L-tryptophan. Notethat VioC and VioD have overlapping function and thus, only VioC wasutilized in the assemblies.

FIG. 44 shows the thermocycler conditions used for the linkedligation/digestion reactions in Example 4.

FIG. 45 shows the results of gel based purification of the pathwayassembly components in Example 4.

EXAMPLES Example 1—2-Part Assembly on Chip

Protocols for on-Chip Assembly

The biology reactions consisted of two 2-part assemblies: RFP or GFPwith a plasmid backbone pSB1C3 (pSB1C3 is also referred to herein as 1C3or 1c3). pSB1C3 encodes resistance to the antibiotic chloramphenicol. Asuccessful assembly of pSB1C3.GFP produces green cells and a successfulassembly of pSB1C3.RFP produces red cells. The number of colonies(yield) and percent of colonies with correct phenotype (efficiency) wasdetermined for test assemblies performed both on chip (using themicrofluidic device) and off chip (in the conventional fashion withtubes and pipettes).

The following parts (nucleic acid sequences), oligos (linkers) andreagents were used, as shown in FIGS. 9 and 10.

-   -   Pre-prepared parts:        -   pSB1C3 DNA pre-digested with EarI        -   RFP DNA pre-digested with EarI        -   GFP DNA pre-digested with EarI    -   uncut pSB1C3.RFP as a positive control for transformation    -   2 pairs of part/linker oligos for the 2-part assemblies, P1, L1,        P2, L2    -   matching biotinylated purification oligos for the above        part/linker oligos, PP1, PL2, PP2, PL2    -   Buffers:        -   Binding buffer: 10 mM Tris-HCl pH 7.5, 500 mM NaCl        -   Elution buffer: 10 mM Tris-HCl pH 7.5    -   Salt solution: 4M NaCl    -   Ligation mix: 15 ul mix is 10 ul T4 DNA ligase buffer, 4 ul T4        DNA ligase (New England Biolabs, MA catalogue #MO202S), 1 ul        EarI (New England Biolabs, MA catalogue #R0528S)    -   Magnetic beads: Streptavidin magnetic beads (New England        Biolabs, MA catalogue # S1420S). These are 1 μm        superparamagnetic particles covalently coupled to a highly pure        form of streptavidin. The magnetic beads bind to the        biotinylated part purification oligos.

The protocol for the on-chip method is as follows and as described inmore detail in FIG. 10:

Ligations: 7.5 uL DNA parts, 0.5 uL of each oligo, 1.5 uL ligase mix

Ligations carried out were pSB1c3 with oligos P1 and L2, RFP with oligosP2 and L1, GFP with oligos P2 and L1

Mix and incubate for 2 hours at room temperature (RT)

The biological and fluidic protocols used for two-step purification ofRFP and pSB1C3 parts were as follows and as described in more detail inFIG. 10 (see part purification step 1 and part purification step 2). Asdescribed below, three different protocols were used and so in FIG. 10,the volume of each reagent is indicated as [Vx], x being a subscriptcorresponding to the particular reagent.

Initial Protocol:

-   -   1. In a new or washed chip, 25 uL of beads are loaded in the        reaction chamber and washed with 50 uL of binding buffer    -   2. Pump 2 uL of oligos with 10 uL binding buffer    -   3. 10 minutes wait at RT    -   4. Wash with 20 uL binding buffer    -   5. 15 uL ligation mix and 2 uL NaCl    -   6. About 10 minutes wait at RT    -   7. Wash with 20 uL elution buffer    -   8. Heat at 65 C then pump another 20 uL elution buffer    -   9. Collect 20 uL elution product e1    -   10. In a new or washed chip, 25 uL of beads are loaded in the        reaction chamber and washed with 50 uL of binding buffer    -   11. Pump 2 uL oligos with 10 uL binding buffer    -   12. 10 minutes wait at RT    -   13. Wash with 20 uL binding buffer    -   14. 20 uL elution product e1 and 2.5 uL NaCl    -   15. 10 minutes wait at RT    -   16. Wash with 20 uL elution buffer    -   17. Heat at 65 C then pump another elution buffer    -   18. Collect 20 uL elution product e2        Updated Purification Protocol A    -   1. In a new or washed chip, 25 uL of beads are loaded in the        reaction chamber and washed with 50 uL of binding buffer    -   2. Pump 5 uL of oligos with 10 uL binding buffer    -   3. 10 minutes wait at RT    -   4. Wash with 20 uL binding buffer    -   5. Pump 15 uL ligation mix and 2 uL NaCl    -   6. About 10 minutes wait at RT    -   7. Wash with 30 uL elution buffer    -   8. Heat at 65 C then pump another 20 uL elution buffer    -   9. Collect 20 uL elution product e1    -   10. In a new or washed chip, 25 uL of beads are loaded in the        reaction chamber and washed with 50 uL of binding buffer    -   11. Pump 5 uL oligos with 10 uL binding buffer    -   12. 10 minutes wait at RT    -   13. Wash with 30 uL binding buffer    -   14. 20 uL elution product e1 and 2.5 uL NaCl    -   15. 10 minutes wait at RT    -   16. Wash with 20 uL elution buffer    -   17. Heat at 65 C then pump another 20 uL elution buffer    -   18. Collect 20 uL elution product e2        Updated Purification Protocol B    -   1. In a new or washed chip, 20 uL of beads are loaded in the        reaction chamber and washed with 50 uL of binding buffer    -   2. Pump 5 uL of oligos with 10 uL binding buffer    -   3. 10 minutes wait at 20 C    -   4. Wash with 50 uL binding buffer    -   5. Pump 15 uL ligation mix and 2 uL NaCl    -   6. About 10 minutes wait at 20 C    -   7. Wash with 50 uL binding buffer    -   8. Pump 5 uL elution buffer, Heat at 65 C then pump another 10        uL elution buffer    -   9. Collect 10 uL elution product e1    -   10. In a new or washed chip, 20 uL of beads are loaded in the        reaction chamber and washed with 50 uL of binding buffer    -   11. Pump 5 uL oligos with 10 uL binding buffer    -   12. 10 minutes wait at RT    -   13. Wash with 50 uL binding buffer    -   14. 10 uL elution product e1 and 2.5 uL NaCl    -   15. 10 minutes wait at RT    -   16. Wash with 20 uL elution buffer    -   17. Pump 5 uL elution buffer in the main chamber, heat at 65 C        then pump another 10 uL elution buffer    -   18. Collect 10 uL elution product e2        Off-Chip/on-Chip Discrepancy:    -   Second wash step only for the off-chip mixture        Pathway assembly: 1.5 uL ligase mix, 4.25 uL plasmid, 4.25 uL        RFP or GFP

Transformation was carried out in competent E. coli cells (New EnglandBiolabs, MA catalogue # C30191) following the manufacturer's protocol.When purification protocol A was followed, the transformation protocolwas: 5 uL ligation in 50 uL cells with 800/900 uLs SOC medium. Whenpurification protocol B was followed, the transformation protocol was: 2uL of ligation in 50 uL cells with 300 uL SOC medium. The transformedcells were plated out onto plates containing the antibioticchloramphenicol and the outcome of the experiments determined bycounting the number of colonies (yield) and percent of colonies withcorrect phenotype (efficiency). A successful assembly of pSB1C3.GFPproduces green cells and a successful assembly of pSB1C3.RFP producesred cells.

Microfluidic Device and System

The method of the present invention is carried out on a microfluidicdevice. In the experiments described in this Example, the biologicalparts, liquids and reagents were first loaded onto the microfluidicchip. Then, an automated control unit platform, based on air andpneumatic valve actuation, allowed the user to perform all thebiological manipulations and reactions on chip. At the end of theprocess, the biological products were then collected from the outputs.

The microfluidic device used in this Example has a number ofmicrofluidic functions. In addition to the basic microfluidic functionsof pumping and mixing, the microfluidic device also has heating andmagnetic control functions. The basic 3D layout of the microfluidicdevice used in this Example is shown in FIG. 12, and FIG. 13 shows thedimensions of the microfluidic device. The device contains two inputchambers, two output chambers (for waste and/or products) and onestorage chamber (temporary holding chamber or reservoir) with fluidchannels linking each of these chambers to a central elliptical reactionchamber. Each “pump” unit for the two input chambers and the storagechamber feature two valves and one pump chamber, as shown in FIG. 13.The storage chamber allows the storage of product whilst flushing thereaction chamber. As can be seen from FIG. 13, the depth of all of thestructures (chambers, channels etc) is 250 μm and the dimensions of themicrofluic device are 70.1 mm×90.1 mm.

As shown in FIG. 13, the inlet channel 1 comprises the first inputchamber, together with its two valves and one pump chamber, the inletchannel 2 comprises the second input chamber, together with its twovalves and one pump chamber, the intermediate product channel comprisesthe storage chamber (temporary holding chamber or reservoir), togetherwith its two valves and one pump chamber, and each of the two wasteand/or product chambers comprises an output chamber together with itsvalve.

A total of 12 pneumatic valves had to be synchronously controlled fordriving the liquid flow in the microfluidic chip to and from thereaction chamber, along with one temperature control for the reactionchamber and one magnetic part for trapping and mixing of the magneticparticles to be used in the biological reactions. The valves can benumbered as shown in FIG. 28.

The microfluidic system used in this Example has three main parts:

-   -   The mechanical assemblies    -   The electronics    -   The software interface        1. The Mechanical Assemblies

The mechanical assemblies can be sub-divided into two separatesub-assemblies. The first one contains both a heating/cooling elementand an electromagnet used to magnetize the magnetic particles in thereaction chamber. FIG. 24 shows isometric left and right views of thissub-assembly.

The peltier device sits on a heat sink which in turn is mounted on topof a cooling fan. The peltier was positioned just underneath thereaction chamber, and was placed just on top of the soft iron part usedto conduct the magnetic field lines to the reaction chamber. The coolingfan slides in a holder in which the base has an array of fourcompression springs. The springs are used to press the peltier elementagainst the chip. All the different components are aligned and fixedusing a set of four mounting brackets (as seen on the left image).

In addition, the first sub-assembly contains an electromagnet which wasused for the magnetic part of the device. The electromagnet also sits ona mounting bracket and has a soft iron bar connected to it. The softiron bar is used as a magnetic canal to provide the chip with therequired magnetic field in order to magnetize the magnetic particles.

As an alternative to the above described heater/cooler module withelectromagnet sub-assembly, another possible configuration would be toput the peltier element on top of the machined soft iron part, justunderneath the reaction chamber, and replace the electromagnet by a hardmagnet which would then allow for both temperature and magnetic controlon chip.

The second sub-assembly is the mechanical structure that holds all thedifferent mechanical components, air connections, electronics, and onwhich the microfluidic device is mounted. The 3D design for thissub-assembly is shown in FIG. 25. The choice of plastic board allowedthe base plate to be manufactured very quickly and efficiently. Thisplate sits in a three parts holder which allows both easy alignment ofthe microfluidic device and enough space for the air connections to comefrom the bottom of the plastic base.

Finally, all the different mechanical parts sit on a thick plastic baseplate, which has all the air connections coming from two opposite sides,and the other two opposites sides have two hook clamps fitted on them.These are used to press the microfluidic device against the plasticboard through the two parts lid.

In the absence of an air connection to the reaction chamber, thevibration of the membrane to promote mixing of the differentbio-products could be induced using a vibrating piezoelectric film thatcan adhere to any physical support underneath, and which would generateslight transversal displacements of the membrane.

2. The Electronics

The liquid flow in the microfluidic channels is driven by synchronizedpumping sequences. These sequences are controlled from a LabVIEWinterface through an electronic platform connected to an array ofpneumatic valves. These valves provide compressed air, through sealedconnections, to the chip thus deforming the elastic membrane coveringthe pumps and valves chambers. By using optimized pumping sequences, itis possible to have accurate control of the liquid flow in themicrochannels. The electronic board that allows controlling each valveseparately, or a set of valves in order to induce pumping sequences, ismainly a relay board made of an array of solid state relays, in whicheach relay is addressing one single pneumatic valve. This electronicboard has two inputs connected to both the DAQ card (with a 25 waycable) and to a power supply, and two outputs connected to the solenoidvalves manifolds by the mean of 15 way cables.

To control the peltier device, two thermocouples were used. The firstone was placed above the Peltier device while the second one was placedbelow the reaction chamber. The electronics behind the temperaturecontrol of the peltier device relies on the use of a programmable powersupply unit, and a National Instruments DAQ card. The Peltier can becontrolled and activated via the LabVIEW control interface describedbelow. The temperature can also be monitored via the same interface.

3 LabVIEW Control Interface

A semi-automated LabVIEW interface was used for the control of themicrofluidic device platform.

Operation of Microfluidic Device for on-Chip Assembly

FIG. 26 summarizes how the biology interacts with the differentmicrofluidic functions for a specific example: the first purification(part purification step 1) of the pSB1C3 ligation mix, as shownschematically in FIGS. 9 and 10 (NB in FIG. 26, pSB1C3 is referred to as1C3). This step and all other purification steps require the entirerange of functions present on the microfluidic system: pumping, mixing,magnetic capture and heating. These steps were extensively demonstratedon-chip (see below).

As can be seen from FIG. 26, the products pumped in were the pSB1C3 DNA,the biotinylated purification oligo PP1 and streptavidin magnetic beads.These products were pumped through to the central reaction chamber wheremagnetic capture of the magnetic beads, mixing of the input products andheating took place. The output of these reactions was the product ofpSB1C3-PP1 bound to the magnetic beads by means of the purificationoligo PP1. The elution product in one of the output chambers waspSB1C3-PP1 whilst the other output chamber contained the waste, whichwas unligated pSB1C3 and PP1.

Pumping to flow liquid from one of the input chambers into the reactionchamber was carried out as shown in FIG. 27. In this Example, only onepump between one input chamber and the reaction chamber was used at atime. Therefore, while one pump between one input chamber and thereaction chamber was activated, the other pump between the other inputchamber and the reaction chamber remained closed at all times.

FIG. 28 summarises a number of different fluidic steps that were used inthis Example and are as described herein.

Magnetic mixing was performed manually, i.e. by moving the magnet abovethe chamber by hand.

Heating was carried out via a Peltier device placed below the chamber,in direct contact with the elastic membrane

Results

Results Obtained with the Updated Purification Protocol A

Four transformations were performed. Table 1 shows the details of thepart ligation and the results obtained. Experiments 3 and 4 representfull off-chip and full on-chip respectively.

TABLE 1 Yield Efficiency (number (coloured/ Name of the ON/OFF CHIP ofn.coloured Experiment transformations status colonies) cells) 1RFP−.1C3+ PART ON-CHIP 42 33.3% 2 RFP+.1C3− PART ON-CHIP 8 37.5% 3RFP−.1C3− OFF-CHIP 8 62.5% 4 RFP+.1C3+ ON-CHIP 102   52%

On the edge on the plate (4) (full on-chip) a 100% efficiency wasreached. This appeared to be due to a concentration effect andtransformation protocol. One reason that explains the ring of cells onthe edge was that the volume of the transformation mixture was too high(850 uL instead of 300 uL): only a small amount of liquid is needed tofill the plate and then the rest of it reaches the edges where it stayshighly concentrated. In the subsequent experiment, only 300 uL oftransformation mixture was used (see below).

Results Obtained with the Updated Purification Protocol B

Four transformations were performed. Table 2 shows the details of thepart ligation and the results obtained. Experiments 3 and 4 representfull off-chip and full on-chip respectively.

TABLE 2 Number of colonies Name of the ON/OFF Number with Experi-transfor- CHIP of phenotypic Efficiency ment mations status colonieschange (%) 1 RFP−.1C3+ PART ON- 14 11 79% CHIP 2 RFP+.1C3− PART ON- 5 480% CHIP 3 RFP−.1C3− OFF-CHIP 12 10 83% 4 RFP+.1C3+ ON-CHIP 7 6 86%

By doubling the washing steps the efficiency of the off-chippurifications was greatly improved. Washing steps to get rid of unwantedoligos seem to be important steps in the on-chip protocol. Thetransformation protocol in these experiments also used a smaller amountof transformation medium.

In conclusion:

-   -   The present inventors have shown that it is possible to carry        out assembly of polynucleic acid sequences on-chip    -   The results obtained on-chip are close to the off-chip results.

Example 2—Chip 3A

A microfluidic device as shown in FIG. 14A was manufactured using cncmachining and tested by washing fluid through the device.

Example 3—5-Part Assembly on Chip

A five-part assembly (RFP, GFP, KanR, AmpR and pSC101) with 0%contamination was demonstrated twice. Two identical experiments werecarried out involving two on-chip tests and two off-chip tests. First ofall the 5 parts, buffer and water were loaded into a chip well, The 5parts and buffer were pumped (˜0.5 uL of each part) sequentially intothe main channel, pushed into the output well and collected with apipette to form the product P1. 1 uL of Water from the water well waspumped into the output channel and pipette off the chip to waste. Morewater and then pumped and formed product P2. P3 was formed by a 5 partsassembly and the addition of 1.5 uL composed equally of blue and yellowfood dye solution and mineral oil. P4 was a conventional off-chip 5 partassembly.

Table 3 summarizes the different products transformed and the resultsobtained.

TABLE 3 Plate number Content Result P1 5 parts ON-CHIP Assembly Assemblysuccessful P2 Water control No contamination P3 5 parts OFF-CHIPAssembly Assembly successful (with added dyes) P4 5 parts OFF-CHIPAssembly Assembly successful

In conclusion, a 5 part assembly without subsequent contamination hasbeen demonstrated (n=2) on chip.

Example 4—10 Part-Assembly on Chip

Violacein Biosynthetic Pathway

Violacein is an industrially-relevant, indole derivative possessinganti-tumoral, anti-ulcerogenic, antitumorigenic, antitrypanosomatid, andantiviral activities. FIG. 43A shows the chemical structure ofViolacein. Biosynthesis of this product can be performed heterologouslyin E. coli and requires only 4 biosynthetic genes. FIG. 43B shows thebiosynthesis of violacein from the precursor, L-tryptophan. Theintensely colored violacein pigment can be used to diagnose successfulassemblies, greatly aiding the assembly debugging process.

10-Part Assembly Design

The 10-way assembly contained the following parts:

TABLE 4 Part Name Part Description Part Number Kan kinase Provides cellswith resistance to the 1076 antibiotic, kanamycin vioA Violaceinbiosynthetic enzyme 1217 vioB-alpha Violacein biosynthetic enzyme, first1471 domain of vioB vioB-beta Violacein biosynthetic enzyme, second 1475domain of vioB vioC Violacein biosynthetic enzyme 1223 vioE Violaceinbiosynthetic enzyme 1225 GFP Includes a promoter and RBS, yields Green1097 Fluorescent Protein P15a origin Allows for plasmid replication bye. coli 1532 (medium copy) RFP Includes a promoter and RBS, yields Red1104 Fluorescent Protein β- Provides cells with resistance to the 1109lactamase antibiotic, ampicillinPart Preparation for High-Order Assemblies

The 10 parts were prepared and then assembled on-chip. Part preparationmethods are described below.

Linked Ligation/Digestion Reaction Conditions

Each part Cloned vector was miniprepped from 4 mL of culture media andeluted in 50 uL EB buffer. Linked ligation-digestion reactions weresetup, using a 40× dilution of T4 DNA ligase and EarI in 1×NEB buffer #2supplemented with 1 mM ATP. Each reaction contained a 25-fold excess ofthe appropriate pre-annealed LOA and POA oligos. Reaction volumes weretypically 90 uL. Reactions were run in a thermo cycler as shown in FIG.44.

Gel-Based Purification of Pathway Assembly Components

65 uL of each reaction was then run on a 1% agarose gel and theappropriate products were extracted from the gel using a Qiagen kit andeluted from the column using 50 uL EB. FIG. 45 shows the gel forfragments 1-7. The dots indicate the extracted band.

Pathway Assembly Off-Chip and on-Chip

Off-Chip Pathway Assembly Steps

A control assembly was conducted off-chip in order to compare to theassembly conducted on chip. The method used was as follows: DNAconcentration of each part was normalized and mixed in the presence of1×NEBuffer #2. Assembly reactions were run for 20 minutes at roomtemperature. 3 uL of the assembly reaction was then used to transformchemically-competent NEB 10 B cells. Transformed cells were recovered inSOC for 1 hour at 37 C before plating on LB/agar/kanamycin plates.

On-Chip Pathway Assembly Steps

The assembly was conducted on-chip using a computer numericallycontrolled (CNC) machined chip.

On-Chip Pathway Assembly Results

Successful assemblies were verified by display of the correct coloredcolony phenotype and the results are shown in Table 5 below. Theoff-chip and on-chip showed essentially equivalent efficiency (88%correct transformants). The total yield was slightly lower on-chip,however the total number of colonies was well above that needed forensuring a successful assembly. The water controls demonstrated thatthere was negligible contamination.

TABLE 5 Plate number Description Results Yield P1 Water control beforeNo contamination — assembly P2 10 parts on chip 88.1% of cells correctlyTotal 84 cells transformed P3 Water control No contamination — afterassembly P4 10 parts off chip 88.2% of cells correctly Total 102 cellstransformed

The invention claimed is:
 1. A method for the assembly of a polynucleic acid sequence from a plurality of nucleic acid sequences in which the polynucleic acid sequence is of a formula N_(n+1), in which N represents a nucleic acid sequence and where n is 1 or greater than 1 and each N may be the same or a different nucleic acid sequence, in which the method comprises: (i) providing a first nucleic acid sequence N1 which has an oligonucleotide linker sequence L1^(3′) attached at the 3′-end of the nucleic acid sequence; (ii) providing a second nucleic acid sequence N2 which optionally has an oligonucleotide linker sequence L2^(3′) attached at the 3′-end of the nucleic acid sequence and which has an oligonucleotide linker sequence L2^(5′) attached at the 5′-end of the nucleic acid sequence, wherein the 5′-end linker sequence L2^(5′) of nucleic acid sequence N2 is complementary to the 3′-end linker sequence L1^(3′) of nucleic acid sequence N1; (iii) optionally providing one or more additional nucleic acid sequences N, wherein nucleic acid sequence N2 has an oligonucleotide linker sequence L2^(3′) attached at the 3′-end of the nucleic acid sequence, and wherein said one or more additional nucleic acid sequences N comprises a terminal additional nucleic acid sequence NZ, and wherein each additional nucleic acid sequence N has an oligonucleotide linker sequence attached at its 3′-end, wherein said terminal additional nucleic acid sequence NZ optionally lacks an oligonucleotide linker sequence at its 3′-end and wherein each additional nucleic acid sequence N has an oligonucleotide linker sequence attached at its 5′-end, wherein for the first additional nucleic acid sequence N3 the 5′-end linker sequence L3^(5′) is complementary to the 3′-end linker sequence L2^(3′) of nucleic acid sequence N2 and for each second and subsequent additional nucleic acid sequence N the 5′-end linker sequence is complementary to the 3′-end linker sequence of the respective preceding additional nucleic acid sequence; (iv) ligating said nucleic acid sequences to form said polynucleic acid sequence, wherein said nucleic acid sequences are optionally purified immediately prior to said ligating; wherein at least step (iv) is carried out on a microfluidic device; and wherein the method does not require polymerase.
 2. A method according to claim 1, wherein said first nucleic acid sequence N1 has an oligonucleotide linker sequence L1^(5′) attached at the 5′-end of the nucleic acid sequence; or wherein said second nucleic acid sequence N2 has an oligonucleotide linker sequence L2^(3′) attached at the 3′-end of the nucleic acid sequence; or wherein said terminal additional nucleic acid sequence NZ has an oligonucleotide linker sequence LZ^(3′) attached at the 3′-end of the nucleic acid sequence; or wherein said first nucleic acid sequence N1 has an oligonucleotide linker sequence L1^(5′) attached at the 5′-end of the nucleic acid sequence, and wherein said second nucleic acid sequence N2 has an oligonucleotide linker sequence L2^(3′) attached at the 3′-end of the nucleic acid sequence, and wherein the 5′-end linker sequence L1^(5′) of nucleic acid sequence N1 is complementary to the 3′-end linker sequence L2^(3′) of nucleic acid sequence N2; or wherein said first nucleic acid sequence N1 has an oligonucleotide linker sequence L1^(5′) attached at the 5′-end of the nucleic acid sequence, and wherein said terminal additional nucleic acid sequence NZ has an oligonucleotide linker sequence LZ^(3′) attached at the 3′-end of the nucleic acid sequence, and wherein the 5′-end linker sequence L1^(5′) of nucleic acid sequence N1 is complementary to the 3′-end linker sequence LZ^(3′) of nucleic acid sequence NZ.
 3. A method according to claim 1, wherein each of said 3′-end linker sequences and said 5′-end linker sequences is partially double stranded; or wherein each said nucleic acid sequence has an overhang at each end.
 4. A method according to claim 3, wherein said overhang at each end of said nucleic acid sequence is produced by digestion with one or more restriction enzymes; or wherein said overhang is 3 or 4 nucleotides in length; or wherein the overhang at the 3′-end of the nucleic acid sequence and/or at the 5′-end of the nucleic acid sequence is the same for each nucleic acid sequence.
 5. A method according to claim 1, wherein each said nucleic acid sequence is attached to its said 3′-end linker sequence and to its said 5′-end linker sequence by ligation.
 6. A method according to claim 1, wherein said nucleic acid sequences are purified on the microfluidic device; or purified using DNA purification spin columns or gel extraction.
 7. A method according to claim 1, wherein each said nucleic acid sequence is a protein coding sequence or a regulatory or control element.
 8. A method according to claim 1, wherein step (iv) is carried out using DNA ligase.
 9. A method for the preparation of a library of polynucleic acid sequences, the method comprising simultaneously producing a plurality of different polynucleic acid sequences using the method according to claim
 1. 